Land and Food Systems, Faculty of
Medicine, Faculty of
Science, Faculty of
Non UBC
Microbiology and Immunology, Department of
Urologic Sciences, Department of
DSpace
Vancouver Prostate Centre
Lin, Zhuohui
Radaeva, Mariia
Cherkasov, Artem
Dong, Xuesen
Tumours develop therapy resistance through complex mechanisms, one of which is that cancer stem cell (CSC) populations within the tumours present self-renewable capability and phenotypical plasticity to endure therapy-induced stress conditions and allow tumour progression to the therapy-resistant state. Developing therapeutic strategies to cope with CSCs requires a thorough understanding of the critical drivers and molecular mechanisms underlying the aforementioned processes. One such hub regulator of stemness is Lin28, an RNA-binding protein. Lin28 blocks the synthesis of let-7, a tumour-suppressor microRNA, and acts as a global regulator of cell differentiation and proliferation. Lin28also targets messenger RNAs and regulates protein translation. In this review, we explain the role of the Lin28/let-7 axis in establishing stemness, epithelial-to-mesenchymal transition, and glucose metabolism reprogramming. We also highlight the role of Lin28 in therapy-resistant prostate cancer progression and discuss the emergence of Lin28-targeted therapeutics and screening methods.
Citation: Lin, Z.; Radaeva, M.;Cherkasov, A.; Dong, X. Lin28Regulates Cancer Cell Stemness forTumour Progression. Cancers 2022, 14,4640. https://doi.org/10.3390/cancers14194640Academic Editor: Kavita ShahReceived: 19 August 2022Accepted: 22 September 2022Published: 24 September 2022Publisher’s Note: MDPI stays neutralwith regard to jurisdictional claims inpublished maps and institutional affil-iations.Copyright: © 2022 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).cancersReviewLin28 Regulates Cancer Cell Stemness for Tumour ProgressionZhuohui Lin 1,2,† , Mariia Radaeva 3,*,† , Artem Cherkasov 3 and Xuesen Dong 31 Department of Microbiology and Immunology, University of British Columbia,Vancouver, BC V6T 1Z4, Canada2 Faculty of Food and Land Systems, University of British Columbia, Vancouver, BC V6T 1Z4, Canada3 The Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia,Vancouver, BC V6H 3Z6, Canada* Correspondence: mradaeva@prostatecentre.com† These authors contributed equally to this work.Simple Summary: Cancer stem cells (CSCs) are a small population of tumour cells bearing stemnesscharacteristics. They have been deemed as a root of cancer development and a promising drug targetfor anticancer therapy. Herein, we discuss the roles of an RNA-binding protein, Lin28, in regulatingcancer cell stemness to drive tumour progression. Lin28 acts on various types of RNAs in cancercells and regulates these RNA functions to control the expression of oncogenes. In these ways, Lin28promotes cancer cell survival, growth, and invasion. We also discuss recent efforts in developingLin28 inhibitors targeting CSCs in tumours.Abstract: Tumours develop therapy resistance through complex mechanisms, one of which is thatcancer stem cell (CSC) populations within the tumours present self-renewable capability and phe-notypical plasticity to endure therapy-induced stress conditions and allow tumour progression tothe therapy-resistant state. Developing therapeutic strategies to cope with CSCs requires a thoroughunderstanding of the critical drivers and molecular mechanisms underlying the aforementionedprocesses. One such hub regulator of stemness is Lin28, an RNA-binding protein. Lin28 blocks thesynthesis of let-7, a tumour-suppressor microRNA, and acts as a global regulator of cell differentiationand proliferation. Lin28also targets messenger RNAs and regulates protein translation. In this re-view, we explain the role of the Lin28/let-7 axis in establishing stemness, epithelial-to-mesenchymaltransition, and glucose metabolism reprogramming. We also highlight the role of Lin28 in therapy-resistant prostate cancer progression and discuss the emergence of Lin28-targeted therapeutics andscreening methods.Keywords: Lin28; let-7; metastasis; metabolism; epithelial-to-mesenchymal transition; prostatecancer; cancer stem cell; Lin28 inhibitors1. IntroductionCancers such as prostate cancer (PC) contain heterogeneous cell populations and, there-fore, respond to anticancer treatments differently [1]. This heterogeneity is reflected not onlyin genomics and transcriptomics but also RNA processing re-programming that can giverise to tumour cells with various co-existing phenotypes and histological presentations [2–4].When a targeted therapy is applied to a tumour, it will be effective in suppressing somepopulations of tumour cells and, at the same time, may provide an opportunity to allowother cell populations to thrive and eventually develop into therapy-resistant tumours. Onegood example is how androgen receptor (AR)-targeted therapy in metastatic PC can resultin short-term tumour suppression effects but also induces castrate-resistant PC (CRPC)in the long term that presents an AR-indifferent phenotype containing weakly expressedAR and AR-regulated PSA, cancer stem cell (CSC) markers (e.g., CD44, CD133, BMI1,EZH2), and even AR negative neuroendocrine PC (NEPC) markers (e.g., CHGA, SYP) [5–7].Cancers 2022, 14, 4640. https://doi.org/10.3390/cancers14194640 https://www.mdpi.com/journal/cancersCancers 2022, 14, 4640 2 of 23Several studies using patient tumour samples, patient-derived xenograft (PDX), and evencell models have demonstrated that there exist landscape switches in phenotypes betweenprimary and therapy-resistant tumour cells, indicating that therapy-resistant tumours areunder the control of different oncogenic signal networks than primary tumours [8–10].These findings underscore that tumour heterogeneity is an inherent challenge for targetedtherapies, and the identification of the driver genes of therapy-resistant tumours couldinform the design of more effective treatments for cancer patients.Cancer stem cells (CSCs) are an important component of tumour heterogeneity andhave been proposed to relate to therapy-resistant tumour progression through two possiblemechanisms. One is that a low number of CSCs pre-exist in primary tumours [11]. Theyare surrounded by a large number of relatively fast-growing non-CSC cells and remain in aquiescence/dormancy state. While therapy-induced stress is efficient to abolish non-CSCs,it can activate CSCs to give rise to tumour cells that are resistant to therapies. Single-cellsequencing of patient PC samples has shown that CRPC cells with characteristic CSCand basal cell phenotypes pre-exist in primary PC [12]. These cells express high levelsof EZH2 and SYP and low levels of AR and AR-regulated PCA3 and have Rb1 loss, allof which are consistent with the molecular profile of classic late-stage NEPC [12]. Ourlab also reported that the NEPC driver gene, SRRM4, is expressed in 16% of hormone-naïve primary tumours, and SRRM4-positive tumour cells increased to ~30% of tumoursunder AR target therapy, supporting the idea that CRPC tumour cells are pre-composite inuntreated PC and become prevalent upon anticancer treatment [13]. The other mechanismis that PC cells with specific genomic features can acquire CSC phenotypes upon exposureto therapy-induced stress [14,15]. Androgen receptor (AR)-mediated signalling is essentialto maintain the luminal epithelial phenotype of prostate adenocarcinoma. However, it hasbeen reported in transgenic mouse models that, when AR inhibitors were used to treat PCcells with TP53 and RB1 gene loss, these cells gained CSC, epithelial-to-mesenchymal, basal,and neuroendocrine phenotypes through the action of transcriptional factors, such as SOX2,and, later, developed into NEPC [15]. In vitro LNCaP PC cells were reported to undergotransdifferentiation to gain CSC phenotypes when treated with AR inhibitors [14]. Gain offunction of SRRM4 in DU145 cells that had TP53 and RB1 gene disruptions, but not LNCaPcells, which were TP53- and RB1-intact, induced a classic CSC gene signature, primarilydriven by the Lin28–SOX2 axis [16]. These findings indicate that prostate adenocarcinomacells are plastic and can alter their phenotypes to cope with therapy-induced stress, duringwhich process the CSC gene network plays a key role.Lin28A and Lin28B (from here referred to as Lin28) are RNA-binding proteins andthe key drivers of CSC phenotypes [17]. Lin28, Oct4, Sox2, and Nanog are the four keyreprogramming factors that induce pluripotency, with Lin28 being the only RNA-bindingprotein [18,19]. Lin28 is weakly expressed in normal differentiated cells but dysregulated incancer cells, resulting in the initiation of a cascade of reactions leading to cancer cells gainingstemness, mobility, and enhanced therapy resistance [20]. Importantly, overexpression ofLin28 downregulates levels of let-7 microRNAs (miRNAs), a class of tumour-suppressormiRNA that regulates a wide range of cancer-related processes, including cell growthand proliferation, epithelial-to-mesenchymal transition (EMT), stemness, and glucosemetabolism reprogramming [20]. Herein, we review the roles of Lin28 in different types ofcancer and discuss the roles of Lin28 in CSC and EMT phenotype development, as well asglucose reprogramming. We also provide an overview of the impact of Lin28 on PC, since ithas emerged as a new diagnostic and therapeutic target of PC [21]. Finally, we summarizeadvances in Lin28 inhibition with small molecules aimed at combating cancer stemnessand metastasis. The hub nature of Lin28 protein makes it an attractive and promising targetfor chemotherapy.Cancers 2022, 14, 4640 3 of 232. Lin28/let-7 axis2.1. Lin28 Structure and FunctionThere are two paralogs of Lin28 protein in vertebrates, Lin28A and Lin28B [22]. Theirstructures share high homology in protein sequences, except that Lin28B has a longerC-terminus containing the nuclear localization signal (NLS) and nucleolar localizationsignal (NoLS) [22,23]. Indeed, various studies have confirmed that the cellular localizationof the two isoforms is different. Lin28B can be localized throughout the cells, even insidethe nucleolus, while Lin28A is predominantly localized at the cytosol [22,23]. Both paralogscontain highly conserved RNA binding regions: the N terminal cold-shock domain (CSD)and a C terminal cysteine–cysteine–histidine–cystine (CCHC)-conserved zinc knuckledomain (ZKD) [22]. Lin28A and B bind the immature forms of let-7 microRNA familymembers and prevent them from being synthesized. Lin28A and Lin28B are mutuallyexclusive in expression in human cancer cell lines [24]. For instance, Piskounova et al. [24]confirmed that only Lin28A was detected in MES and IGROVE1, while only Lin28B wasdetected in HEK293, H1299, HepG2, and K562 cell lines. However, a minority of cancers,including ovarian cancer, germ cell tumours, and teratomas, express both paralogs ofLin28 [25–27].2.2. Let-7 BiogenesisLet-7 miRNAs are key regulators of embryonic development and cancer progression.They are synthesized through several steps (Figure 1). First, the primary miRNA transcriptsof let-7s (pri-let-7s) are transcribed from the MIRLET7 genes by RNA polymerase II [28,29].These transcripts form structures of hairpin stem loops consisting of a stem region, andthey have a characteristic pre-element (preE) bulge and a preE loop [29,30]. Second,pri-let-7 is cleaved at the stem-loop structure of the preE by Drosha (a nuclear-localizedRNase III) and Pasha (double-stranded RNA binding protein) into a shorter (60–80 nts)pre-let-7 hairpin structure [29]. The pre-miRNA is then exported out of the nucleus byexportin-5 [31]. Finally, the preE element of pre-let-7 is cleaved by Dicer to produce amature let-7 miRNA duplex with a 3’ overhang in the cytoplasm [30]. Some members ofthe let-7 family (pre-let-7a-2, -7c, and -7e) contain a bulged adenosine/uridine residue atthe 3’ end that impairs them from being recognized by cytoplasmic Dicer complex [32,33].In these cases, a terminal uridylyl transferase (TUT2, TUT4, or TUT7) will mono-uridylatethe 3’ overhang of pre-let-7 to allow Dicer cleavage [32–34]. At the cytoplasm, HIV-1TAR RNA-binding protein (TRBP) interacts with Dicer and recruits argonaute protein (i.e.,AGO2) to form RNA-induced silencing complex (RISC) [35]. One strand of the mature let-7duplex is preferentially incorporated into the active parts of the RISC [23,36]. Besides theconventional described maturation process, Drosha/Dicer-independent biogenesis of let-7microRNA also exists [37,38].2.3. Lin28 Regulates let-7 MaturationAs shown in Figure 1, two paralogs of Lin28 bind to let-7 to prevent it from beingfurther processed [39,40]. The two conserved domains, CSD and ZKD, have a high affinityto let-7 precursors at the terminal loop region of preE [30]. Specifically, ZKD binds to the 3’end of the preE bulge containing the GGAG motif, while CSD preferentially binds to thepreE loop with the NGNGAYNNN motif [30]. Mayr et al. [41] showed that CSD binds to mi-croRNA first and then induces a conformational change in the preE, making it available forZKD binding. Although CSD has a higher affinity to miRNA, ZKD binding to the sequenceis required and sufficient to recruit TUTase enzymes and induce oligouridylation [42]. Inthe cytosol, the Lin28B CSD recognizes the preE loop of pre-let-7 while allowing ZKD tobind with the conserved motif preE bulge within let-7 (GGAG) [22,32]. ZKD binding tomiRNA blocks the Dicer cleavage site, which is proximal to the GGAG-conserved preEbulge, and prevents pre-let-7 maturation [22]. It is worth mentioning that some members ofthe let-7 family can escape Lin28-mediated inhibition [43]. For instance, Triboulet et al. [43]confirmed that human let-7a-3 and its murine ortholog, mouse let-7c-2, bypass Lin28A-Cancers 2022, 14, 4640 4 of 23mediated repression. However, these pathways must be less common because most of thestudies confirm that let-7 levels are tightly regulated by Lin28A and Lin28B.Lin28A and Lin28B inhibit let-7 maturation in distinct cellular compartments [40].Lin28A is mostly found in the cytosol while Lin28B is mostly found in the nucleus, asLin28B has an NLS [24]. In the nucleus, RNA-binding protein Musashi (MSI1) inducesLin28B nuclear localization and aids ZKD recognition of the terminal loop of pri-let-7,inhibiting Drosha cleavage [40,44]. Despite the different localization of Lin28A and B incells, some other studies have proposed that the localization of the Lin28A and B paralogsmight be cell cycle-dependent [45]. Furthermore, Piskounova et al. [24] found that, whileLin28A requires TUTase to inhibit let-7 maturation, Lin28B acts in a TUTase-independentmanner, recruiting DGCR8 microprocessor instead.Cancers 2022, 14, x 4 of 25 Figure 1. The biogenesis of let-7 microRNA. In the absence of Lin28B, pri-let-7s are cleaved into pre-let-7 miRNAs and then converted to mature let-7 with the aid of Dicer in both cytoplasm and nu-cleus. The stages at which Lin28A and/or B inhibit let-7 maturation are marked. The lower right square shows the structure of pri-let-7. The upper right square demonstrates the site where Dicer cleaves pre-let-7. The left square shows the bipartite binding of Lin28A/B to pri/pre-let-7 members. Created with BioRender.com. 2.3. Lin28 Regulates let-7 Maturation As shown in Figure 1, two paralogs of Lin28 bind to let-7 to prevent it from being further processed [39,40]. The two conserved domains, CSD and ZKD, have a high affinity to let-7 precursors at the terminal loop region of preE [30]. Specifically, ZKD binds to the 3’ end of the preE bulge containing the GGAG motif, while CSD preferentially binds to the preE loop with the NGNGAYNNN motif [30]. Mayr et al. [41] showed that CSD binds to microRNA first and then induces a conformational change in the preE, making it avail-able for ZKD binding. Although CSD has a higher affinity to miRNA, ZKD binding to the sequence is required and sufficient to recruit TUTase enzymes and induce oligouridyla-tion [42]. In the cytosol, the Lin28B CSD recognizes the preE loop of pre-let-7 while allow-ing ZKD to bind with the conserved motif preE bulge within let-7 (GGAG) [22,32]. ZKD binding to miRNA blocks the Dicer cleavage site, which is proximal to the GGAG-con-served preE bulge, and prevents pre-let-7 maturation [22]. It is worth mentioning that some members of the let-7 family can escape Lin28-mediated inhibition [43]. For instance, Triboulet et al. [43] confirmed that human let-7a-3 and its murine ortholog, mouse let-7c-2, bypass Lin28A-mediated repression. However, these pathways must be less common because most of the studies confirm that let-7 levels are tightly regulated by Lin28A and Lin28B. Lin28A and Lin28B inhibit let-7 maturation in distinct cellular compartments [40]. Lin28A is mostly found in the cytosol while Lin28B is mostly found in the nucleus, as Lin28B has an NLS [Error! Reference source not found.]. In the nucleus, RNA-binding protein Musashi (MSI1) induces Lin28B nuclear localization and aids ZKD recognition of the terminal loop of pri-let-7, inhibiting Drosha cleavage [40,44]. Despite the different lo-calization of Lin28A and B in cells, some other studies have proposed that the localization Figure 1. The biogenesis of let-7 microRNA. In the absence of Lin28B, pri-let-7s are cleaved intopre-let-7 miRNAs and then converted to mature let-7 with the aid of Dicer in both cytoplasm andnucleus. The stages at which Lin28A and/or B inhibit let-7 maturation are marked. The lower rightsquare shows the structure of pri-let-7. The upper right square demonstrates the site where Dicercleaves pre-let-7. The left square shows the bipartite binding of Lin28A/B to pri/pre-let-7 members.Created with BioRender.com.3. The Role of Lin28 in CancersThe roles of Lin28A and Lin28B in cancer development and progression have beenelucidated over the last decade. Numerous studies have found that overexpression ofLin28A/B leads to reduced levels of the let-7 members that stimulate tumour cells to gainstemness and invasiveness. Not surprisingly, clinical studies have confirmed that Lin28overexpression correlates with higher chances of metastasis and overall poor survival rates,and we have summarized the main findings on Lin28 in various cancer types in Table 1.The broad range of cancer types and cancer cell lines that rely on Lin28/let-7 regulationindicates a central role for the pathway in cancer biology. Indeed, a comprehensive analysisof Lin28A and Lin28B expression over a panel of 527 human cancer cell lines revealedthat the paralogs are overexpressed in roughly 15% of cancer types [25]. The plethora ofLin28A/B functions is explained by the abundance of downstream genes, including directtargets of let-7, Sox2, HMGA2, and c-Myc [17]. Furthermore, various studies have shownCancers 2022, 14, 4640 5 of 23that therapeutic inhibition, gene knockout or RNA silencing of Lin28 reverse the stemnessand invasive phenotypes (Table 1), highlighting that aberrant Lin28 expression is functionalin the control of tumour cell aggressiveness. Additionally, overexpression of Lin28B intransgenic murine models was sufficient to induce hepatoblastoma and hepatocellularcarcinoma, while deletion or silencing of Lin28B gene prolonged survival [46]. Thesestudies collectively demonstrate the key importance of Lin28A/B in the development andprogression of cancers.Table 1. Lin28 roles in cancers. Lin28A and B expression in human cancer cell lines were confirmedfrom the Cancer Cell Line Encyclopedia (CCLC) Dependency Map database.PrimaryBody Site Tumour Name Samples StudiedLin28A or BExpression Results Ref.LungNon-Small celllung cancer(NSCLC)Lung tissuesCell lines: A549,NHBEA549: bothNHBE: n/a(1) Lin28B and metabolic enzymeglycine decarboxylase (GLDC) ↑ intumour-initiating cancer cells (TICs)are necessary and sufficient to inducetumour sphere formation[47]Small cell lungcancer (SCLC)Cell lines:NCI-H446 NCI-H446: both(1) Lin28A ↑ with c-Myc ↑ causespri-let-7 ↓and mature let-7 ↓(2) Lin28A inhibition triggers cellcycle arrest and ↓ cell growth(3) Lin28B LoF results in CDC25A ↓,an activator of G1 to S phasetransformation, thus preventingcell division[48]BrainGlioblastomamultiforme (GBM)Glioma tissuesCell lines: U251,U373All cell lines: Lin28A(1) Lin28A ↑ leads to poor diagnosisand ↓ survival rate(2) Lin28A ↓ induces cell cycle arrestin G1 phase, ↑ apoptosis, and ↓colony count[49]Atypical teratoidrhabdoid tumour(AT/RT)AT/RT primarytumour tissuesCell lines:BT12/37,CHLA-06/04/06All cell lines: both(1) High levels of Lin28A and Lin28BmRNA coupled with low levels oflet-7 in the majority of tumours(2) Lin28A KO ↑ let-7 mRNA,downregulates KRAS mRNA,reduces cell growth and proliferation,↓ clonogenicity, and inducesapoptosis in cells(3) Lin28A ↓ in xenograft results in atwo-fold ↑ in median survival rate[50]ProstateAndrogen-independentprostate cancer(AIPC)Cell lines: VCaP,LNCaP, PC3,Du145VCap: bothLNCap: Lin28BPC3: Lin28BDu145: Lin28B(1) Lin28B positively correlates withc-Myc protein but not mRNA level(2) Lin28B negatively correlates withmiR-212, a tumour suppressor(3) Computational predictionssuggest that c-Myc protein activatesLin28B, which in turn ↓miR212miRNA activity[51]Therapy-inducedneuroendocrineprostate cancer(t-NEPC)Cell lines: LNCaP,C4-2, RWPE-1,22RV1, PC-3, VCaP,Du145, NCI-H660/H82/H69,LASCPCLNCap: Lin28BC4-2: n/aRWPE-1: n/a22RV1: n/aPC-3: n/aVCaP: bothDu145: Lin28BNCL-H660/H82/H69:Lin28BLASCPC: Lin28B(1) Lin28B ↑ and Sox2 ↑mRNAs andprotein levels in tumours, xenografts,and cells(2) Let-7d ↓ results in HMGA2 ↑and Sox2 ↑(3) Lin28B induces cancer stem celland neuroendocrine biomarkers[16]Cancers 2022, 14, 4640 6 of 23Table 1. Cont.PrimaryBody Site Tumour Name Samples StudiedLin28A or BExpression Results Ref.ProstateProstate cancer(PC)Cell lines: PrEC,RPWE-1, LNCaP,Du145, PC3All cell lines: both(1) ESE3/EHF binds Lin28A and Bpromoter, deactivating its expressionand, thereby, ↑ let-7 miRNA levels(2) Lin28A and B inhibition resemblesthe effects of ESE3/EHF, as it ↓ CSCphenotype[52]GastrointestinalColon carcinomaColon cancertissuesCell lines: SW480,HCT116All cell lines: both(1) Lin28B ↑ in colon cancer tissues ↑metastasis and correlates with poorpatient survival rate(2) Lin28B inhibition leads to ↓migration and ↑ drug-inducedcytotoxicity in cells[53]ColonadenocarcinomaColon carcinomatissuesCell lines: DLD-1,LoVoDLD-1: Lin28BLoVo: both(1) Lin28B ↑ reduces patient survival,↑ tumour resistance and chancesof relapse(2) Lin28B ↑ LGR4 and PROM1colonic cancer cell markers in vivo[54]Gastricadenocarcinoma(GAC)Gastric cancertissues Lin28B(1) Lin28B ↑ in GAC tissues(2) Lin28B expression level ispositively correlated withclinicopathological parameters ofGAC patients (i.e., lymph node status,TNM, tumour invasion, and GAC celldifferentiation) and negativelycorrelated with survival rates[55]Colorectal cancer(CRC)ColonadenocarcinomatissuesBoth(1) Lin28A/B levels are not related toliver metastasis development(2) Lin28A/B expression is negativelycorrelated with Sox2 expression[56]Wnt-activatedesophagealsquamous cellcarcinoma (ESCC)ESCC tissuesCell lines: TE-1,ECA109, KYSE-150TE-1: bothECA109: Lin28AKYSE-150: both(1) Let-7a ↓correlates with metastasisand cancer recurrence(2) Let-7a-mimic and Lin28A/Bsilencing reduce EMTLet-7a ↓ reduces protein levels ofEMT divers Snail and SLUG(3) Wnt/β-catenin ↑ increases theexpression of its Lin28A/B[57]Oral squamous cellcarcinoma (OSCC)OSCC tissuesCell lines: SCC9,SCC15, SCC25SCC9: bothSCC15: bothSCC25: Lin28B(1) Lin28B expression correlates withearlier disease recurrence andpromotes cancer cell migration(2) Lin28B, but not Lin28A, isassociated with poorpatient prognosis(3) Lin28B ↑migration, invasion,proliferation, and clonogenicity(4) Lin28B ↑ IL-6, HMGA2, Snail,Twist, VEGF, and Survivin expression[58]KidneyRenal cellcarcinoma (RCC)Nephrectomytissues Lin28A (1) Lin28A expression is normal [59]Wilms’ tumour Kidney tissues ofmice embryoEmbryonic stem cells:both(1) Lin28A and B ↑ blocksdifferentiation of embryonic kidneycells, transforming them intonephrogenic progenitors that initiateWilms tumour(2) Lin28B/let-7 pathway promotesglomerulus-like structuretumour formation[60]Cancers 2022, 14, 4640 7 of 23Table 1. Cont.PrimaryBody Site Tumour Name Samples StudiedLin28A or BExpression Results Ref.CirculationMixed-lineageleukemia (MLL)Bone marrowtissueMLL-AF6/9/10/ENL/Gas7Lin28B(1) Lin28B ↑ due to c-Myc ↑(2) Lin28B inhibits let-7a/g and ↑tumour growth(3) Let-7 levels’ restoration results indifferentiation of leukemia blasts[61]Acute myeloidleukemia (AML)AML xenograftmice modelAML cell linesLin28A and B(1) A small molecule inhibitor (1638)of Lin28 ↓ cell growth andclonogenicity(2) In xenograft models, 1638 leads to↑ let-7 levels, ↓ LCS counts andtumour growth[62]Juvenilemyelomonocyticleukemia (JMML)subtypeJMML tissues Lin28B(1) Lin28B is a characteristic of anovel molecular subgroup of JMML(2) Lin28B correlates with HbF, aprognostic JMML marker(3) Lin28B is a marker ofpoor survival[63]LiverHepatocellularcarcinoma (HCC)HCC tissuesCell lines: Hek293,Cos-7 HepG2/3B,Li-7, HLE, Huh6/7,MCF-7All cell lines: Lin28BHuh6/7: both(1) First report of Lin28B(2) Lin28B ↑ in HCC cell lines andtissue samples(3) Lin28B localizes in the cytoplasm(4) Lin28B lacking CSD domain isexpressed in normal liver cells[45]Cell lines: HeLa,HepG2, Huh7,Hep3BHeLa: Lin28BHepG2: bothHuh7: bothHep3B: both(1) Lin28B ↓ let-7 biogenesis byinhibiting its maturation through 3’uridylation that blocksDicer processing[32]Hepatoblastoma(HB) Mouse models Lin28B(1) Lin28B alone is sufficient to induceliver tumourigenesis in mice model(2) Lin28B ↑ in Myc-drivenmouse models(3) Lin28B directly ↑ an lgf2 mRNAbinding protein that ↑ tumour growth[46]OvarianOvarian carcinoma A panel of 527human Both(1) Lin28A and Lin28B ↑ in ovariancarcinoma histological grade 2 or 3 [25]Epithelial ovariancancerEpithelial ovariancancer patienttissuesBoth(1) Lin28B ↑ in patients leads to shortprogression-free and lowsurvival rates(2) Lin28B blocks let-7a maturationand positively correlates withpri/pre-let-7a(3) A positive correlation betweenLin28B and insulin-like growth factorII (IGF-II) may contribute to theadverse effect in ovarian cancer[64]Ovarian primitivegerm cell tumours(GCTs)Ovarian cancertumour tissues Both(1) Lin28A ↑ significantly contributesto primitive ovarian GCTs (primaryand metastatic dysgerminomas,gonadoblastoma, Yolk Sac tumour,and embryonal carcinoma), immatureteratomas, and neuroepithelial tissues(2) Lin28A can be used as adiagnostic marker for GCTs[26]Cancers 2022, 14, 4640 8 of 23Table 1. Cont.PrimaryBody Site Tumour Name Samples StudiedLin28A or BExpression Results Ref.TesticleTesticular germ celltumours (GCTs)Testicular cancertumour tissues Both(1) Lin28A and Lin28B are stronglydetected and have specificity inmetastatic testicular GCTs, includingclassic seminoma, embryonalcarcinoma, and yolk sac tumour(2) Lin28A, Lin28B, and SALL4 act assensitive markers for extragonadalyolk sac tumours[27]BreastBreast cancersBreast cancertissuesHB22, T47D,MCF7, Bcap-37,SK-BR-3, MDA-231HB22: n/aT47D: bothMCF7: Lin28ABcap-37:SK-BR-3: Lin28AMDA-231: n/a(1) Lin28A/B ↑ confers resistanceto paclitaxel(2) Lin28A/B KO causes ↑ sensitivityto paclitaxel in T47D cell line(3) Lin28A/B ↑ in breast cancertissues of relapsed cancer(4) Lin28A/B induces p21 andRb expression[65]Triple-negativebreast cancersubtypePrimary breastcancer tissue4TO7,MDA-MB-231,293 TAll cell lines: Lin28B(1) Lin28B ↑ pre-metastatic genes inlung tissues, facilitating neutrophilaccumulation, N2 conversion, and thedevelopment of the immune-suppressive pre-metastatic niche(2) Let-7s exosomes ↓ inpre-metastatic niches ↑ IL-6 and IL-10,contributing to the incidence of lungmetastasis and increased tumour size(3) Lin28B and let-7s are markers forpoor survival and lung metastasis[66]Breast cancerMouse modelsMDA-MB-231,MCF10A, bone(1833) and lung(4175) metastaticbreast cancer cellsMDA-MB-231: Lin28BMCF10A: both1833: both4175: both(1) The dysfunction of Raf kinaseinhibitor protein (RKIP), a metastasissuppressor, facilitates Myc binding tothe Lin28A and B promoter(2) Lin28A and B suppression leads to↑ let-7, inhibits HMGA2 (an activatorof metastatic genes), and ↓ bonemetastasis[67]LymphNodePapillary thyroidcarcinoma (PTC) PTC tissues Both(1) Lin28A and B was expressed in40.5% of PTC samples(2) Patients with Lin28A and Bexpression had larger tumour size,more frequent lymph nodemetastasis, and aggressive tumourcharacteristics(3) Lin28A and B serves as aprognostic marker in PTC[68]SpinalCordMYCN-amplifiedneuroblastomaCHP-212,SK-N-AS, SY5Y,BE2C, KellyCHP-212: bothSK-N-AS: Lin28BSY5Y: Lin28BBE2C: Lin28BKelly: Lin28B(1) Lin28B ↑ cell migration in vitroand metastatic abilities in vivo(2) Lin28B KO ↓ tumour size inmice xenograft(3) Lin28B mediates liver metastasisMYCN amplification significantlyinduces Lin28B expression[69]BladderBladder cancer(BLCa)BLCa tissuesCell lines: T24,UM-UC-3, J82,SV-HUC-1All cell lines: both(1) LINC01451 IncRNA directly bindsto the promoters of Lin28A andLin28B in tumour tissues(2) LINC01451 ↑ EMT by ↓biomarkers of TGF-β/Smadsignalling pathways throughLin28A/B regulation[70]↑—increase; ↓—decreaseCancers 2022, 14, 4640 9 of 234. The Role of Lin28 in Regulating the CSC PhenotypeEarly evidence of the key role of the Lin28/let-7 axis in the development of the CSCphenotype was presented by Yu et al. [71] in a study on breast cancers. They postulatedthat low levels of cellular let-7 result in upregulation of HMGA2, a suppressor of differenti-ation, and H-RAS, an enhancer of self-renewal ability [71]. They showed that an artificialupregulation of let-7 through lentiviral infection reduced the regenerative abilities of undif-ferentiated cells and, subsequently, prevented tumour growth and metastasis [71]. Similarfindings were reported for pancreatic cancer, where low levels of let-7 were found to beresponsible for the upregulation of pluripotency genes, including Sox2, Sox9, Oct4, Nanog,and c-Myc [72]. Further evidence supporting the role of Lin28/let-7 in the development oftreatment resistance through the CSC phenotype was demonstrated in breast and gastriccancer [71,73]. Overall, the Lin28/let-7 axis plays an important role in cancer stemness,and an understanding of mechanisms underlying Lin28-mediated CSC initiation mightilluminate some important targets for metastatic and therapy-resistant cancer treatment.Several known let-7-regulated signalling pathways are implicated in CSC develop-ment, including the Wnt/β-catenin, NOTCH/hedgehog, MAPK/ERK, PI3K/AKT, andSTAT3/NFκB/cytokines pathways [74]. A significant amount of research on breast cancerprogression linked Lin28/let-7 with the development of CSC phenotypes through the afore-mentioned signalling pathways. For example, breast cancer has been shown to respondto tamoxifen treatment and radiotherapy more effectively as a result of let-7c/d-inducedsuppression of the Wnt pathway, which stimulates CSC renewal and proliferative abili-ties [75,76]. Similarly, the CSC phenotype in breast cancer cells was suppressed after theattenuation of Lin28A expression caused let-7b-dependent Wnt pathway inhibition [77].Besides breast cancer, the Wnt signalling suppressed by let-7 was shown to inhibit EMTand self-renewal phenotypes in CSCs of lung and esophageal cancers, as well as in hepato-cellular carcinoma [77–79]. The importance of the Lin28/let-7 axis on Wnt signalling wasalso highlighted in a study of Wnt-activated esophageal squamous cell carcinoma (ESCC)clinical tissues. A clear correlation between metastasis, migration, and invasion with thelet-7 members was observed [57].The NOTCH/hedgehog pathway has also been indirectly linked to the stemnessphenotype and metastatic abilities of cancer cells. For instance, sonic hedgehog protein,along with CSC cell surface markers, was found to be upregulated in highly metastaticpancreatic cancer [80]. Such upregulation was accompanied by low levels of let-7 members,which were shown to directly affect sonic hedgehog expression [81]. In non-small lungcancer cells (NSCLC), upregulation of let-7c, along with miR-200b, coupled with inhibitionof the Hh signalling pathway resulted in a reduction of CSC markers and EMT phenotype,as well as re-sensitization to chemotherapy [82].Aberrant regulation of the STAT3/NFkB pathway, which promotes mesenchymaland CSC phenotypes, is also related to the Lin28/let-7 axis. For example, in NSCLC,NFkB binds to Lin28B chromatin, leading to overexpression of Lin28B and subsequentsuppression of let-7 [83]. Similarly, STAT3 directly stimulates Lin28A/B expression bybinding to its promoter in breast cancer cells [84]. In both studies, NFkB- and STAT3-mediated activations of Lin28 enhanced EMT and CSC phenotypes. STAT3/NFkB pathwayis stimulated by pro-inflammatory cytokines produced by M1 macrophages, thus mediatingan immune-dependent activation of the Lin28/let-7 axis [85]. Other studies also reportedthat IL6 cytokines create a positive feedback loop in Lin28/let-7 axis activation throughstimulation of NFkB, leading to the transformation of breast epithelial cells into CSCs.Overall, the STAT3/NFkB pathway has been found to deliver immune signals down to theLin28/let-7 axis.Cancers 2022, 14, 4640 10 of 235. The Role of Lin28 in EMT and MetastasisEMT, the initiation step of invasion and metastasis, is defined by the destabilizationof the adherent and tight junctions that maintain the integrity and apical-basal polarityof epithelia [86,87]. The transformation of the epithelial cells into mesenchymal cells is acharacteristic change when tumour cells gain an invasive phenotype and obtain motilityto progress into more malignant tumours [86,87]. The epithelial cell adherent junctionconsists of cadherin adhesion receptors, such as N cadherin and E cadherin, which areresponsible for calcium-dependent adhesion and maintenance of epithelial cell plasticity,respectively [87]. During EMT, cells lose E cadherin but gain N cadherin expression. EMTis also accompanied by several other phenotypic changes in epithelial cells, includingcytoskeleton dynamics, which allow cancer cells to gain high mobility [87].5.1. Lin28 Promotes EMT and Metastasis in let-7-Dependent MannerLin28 is one of the well-studied EMT-inducing factors [19]. Clinical evidence hasshown that Lin28A and B are correlated with cancer cell invasion and migration. Earlyresearch first marked low levels of let-7 family members as a prerequisite for metastasisand one of the key drivers of mesenchymal phenotype switch in epithelial cells [88–91].The discussed mechanisms involved loss of let-7-regulated suppression and subsequentupregulation of genes, such as E-cadherin transcriptional repressors ZEB1 and SIP1 in abreast cancer model, GAB2 and FN1 in mammary carcinoma, and developmental genesTwist and Snail in an oral squamous cell carcinoma (OSCC) model. Importantly, the key roleof let-7 in EMT was further proven by reversing the EMT process through the restorationof let-7 levels [90,92]. These findings suggest that Lin28A and B are both direct negativeregulators of let-7 in initiating EMT.The direct role of Lin28A/B in the development of EMT through let-7 regulation hasalso been experimentally demonstrated by a series of studies. Lin28A/B was shown to besignificantly upregulated in mesenchymal type cells (MDA-MB-231 and SK-3rd) comparedto epithelial type cells (MCF-7 and BT-474) [20]. Furthermore, Lin28A/B was found tobe responsible for higher colony formation rates and migratory abilities in breast cancercells. In triple-negative breast cancer (TNCB) cells, overexpression of Lin28A/B lowersthe level of E cadherin while increasing N cadherin, thereby evidently contributing toEMT development and conferring cancer stem cell characteristics [19]. Similarly, over-expression of Lin28B in TNCB was found to promote lung and breast cancer metastasisthrough the development of an immune-suppressive pre-metastatic niche marked by neu-trophil recruitment and subsequent conversion of N2 phenotype [66]. In particular, Lin28Bwas shown to increase IL-6 and IL-10 cytokine production to polarize neutrophils intometastatic-promoting N2 phenotype-inhibiting T cells, which normally prevent malignantcell migration [66]. These studies collectively demonstrate that Lin28 initiates EMT andmetastasis in breast cancer models not only by causing cell–cell adhesion loss but also byacting as an immune-suppressive agent.Another study emphasized the role of Lin28 in EMT and metastasis by discoveringLin28 as a target of a known metastasis suppressor in PC, called Raf kinase inhibitoryprotein (RKIP) [67]. The study showed that RKIP indirectly suppresses Lin28A/B throughmitogen-activated protein kinases (MAPK) and Myc [67]. The reduction of Lin28A/Bconsequently restores let-7 levels, which in turn block the expression of a metastasis-activating gene HMGA2 [67]. The dysregulation of RKIP in breast cancer cells was linkedto breast cancer development and invasion through the reversion of the aforementionedcascade involving HMGA2-let-7-Lin28A/B-Myc-MAPK-RKIP [67]. Importantly, whilemost of the studies implicated the role of Lin28 in cancers by showing the downstreameffects of Lin28, this study proved its significance by showing that Lin28 is a direct upstreamregulator of RKIP.Cancers 2022, 14, 4640 11 of 235.2. Lin28 Promotes EMT and Metastasis in let-7-Independent MannerLin28 can also regulate cell invasion and migration independently of let-7. For example,Lin28A/B was found to act as a post-translational driver of metastatic phenotype viadirect enhancement of the translation of MYCN-induced transcripts in MYCN-amplifiedneuroblastoma [69]. In this study, ZKD of Lin28B bound the RPS29 protein, which wasshown to correlate with low patent survival, cause cell cycle arrest, and induce cancer cellmorphological differentiation. Importantly, the authors showed that the loss of Lin28Breduced the metastatic potential of the tumour cells, leading to a complete reversal of stage4 to stage 1 of neuroblastoma. This let-7-independent function of Lin28 demonstrates thatLin28 has a broad range of effectors that are highly clinically relevant.6. The Role of Lin28 in Glucose MetabolismLin28 plays a critical role in the Warburg effect, which is defined as the reprogrammingof glucose metabolism to enhance tumourigenesis [93]. Cancer cells predominantly rely onaerobic glycolysis and utilize glucose as the energy source instead of more efficient defaultpathways, such as the citric acid cycle and oxidative phosphorylation in mitochondria [93].The shifts in glucose metabolism provide additional plasticity for cancer cells and evenenhance therapy resistance [94]. Cancer cells require higher energy demands, not onlybecause of the increased replication and proliferation rates but also hostile tumour microen-vironments, such as changes in pH. Thus the cancer cells’ metabolic plasticity becomescritical for survival. This is even more true for cells that undergo EMT and metastasis,during which processes the cells experience higher oxidative stress and have to alter theirmetabolism to withstand the hurdles of metastatic spread [95].Early evidence showed that the Lin28/let-7 axis regulates metabolism through pyru-vate dehydrogenase kinase 1 (PDK1) in an oxygen- or hypoxia-inducible factor-1 (HIF-1)-independent manner [96]. Lin28B was found to control the aerobic glycolysis metabolicstate and lower the microenvironment pH through the Lin28B/Myc/miR-34a-5p pathwayin breast cancer stem cells (BCSCs) [97]. Lin28B-positive MDA-MB-231 and H1299 cells useda significant amount of glucose for aerobic glycolysis and produced a high concentration oflactate, while Lin28B-negative MDA-MB-231 cells had significantly lower rates of glucoseuptake and lactate secretion [97]. The high level of lactate in these BSCSs promoted a lowpH microenvironment that enhanced stemness properties. Furthermore, low pH increasedthe abundance of cells expressing high levels of aldehyde dehydrogenase 1 (ALDH1), aCSC biomarker [97]. Myc and Lin28B proteins were also significantly overexpressed inspheroid cells and ALDH-overexpressed cells under a low pH environment [97]. Moreover,the biogenesis of miR-34a-4p, a Myc-suppressed miRNA, is downregulated by Lin28B [97].Lin28B activates Myc expression, which, in turn, blocks miR-34a-5p to induce glycolysisand inhibits oxidative phosphorylation in BCSCs. Lin28B inhibition by a small moleculeC902 reduced Myc expression and lactate secretion in BCSCs. This thereby reversed pH toclose to neutral and restored miR-34a-5p levels, which led to reductions in tumour spheroidformation, BCSC migration capability, and tumour sizes. Moreover, the blockade of theLin28/Myc/miR-34a-5p axis successfully hampered pulmonary metastasis of BCSC inin vitro models [97]. Based on these findings, this study concluded that BCSC relies onaerobic glycolysis activated through the Lin28B/Myc/miR-34a-5p pathway.Similarly, another study determined the role of the Lin28B/let-7 pathway in regulatingglycolytic metabolism, ATP production, and neurosphere formation in neuroblastoma(NB) [98]. The researchers proposed a pathway whereby ornithine decarboxylase (ODC)regulates polyamine production to induce hypusination of eIF-5A, which in turn regulatesthe Lin28B/let-7 pathway [98]. The study showed that ODC inhibition by difluoromethy-lornithine (DFMO) restores ATP levels, let-7 expression, and glycolytic metabolism invarious degrees depending on the NB cell lines (BE(2)-C, SMS-KCNR, and CHLA90) [98].Lin28B and MYCN protein expression and ATP levels significantly decreased in BE(2)-Cand SMS-KCNR cell lines in response to DFMO treatment [98]. Furthermore, as let-7expression increased upon DFMO treatment, there was a decrease in glycolytic metabolismCancers 2022, 14, 4640 12 of 23in vivo [98]. Therefore, these researchers concluded that Lin28 significantly contributes toglucose metabolism reprogramming in NB cell lines.Although one of the main known roles of Lin28 is to promote tumourigenesis andmetabolic diseases, the reprogramming of glucose metabolism by Lin28A/B brings ad-vantages to the resistance to diabetes, skeletal muscle maintenance, and acceleration oftissue repair [99–101]. For instance, overexpression of Lin28A and Lin28B leads to aninsulin-sensitized state, which subsequently improves glucose tolerance in Lin28A/B ge-netically modified aging mice. It allowed the mice to gain resistance to diet-induceddiabetes under high-fat diet conditions [99]. Another study showed that Lin28A knockouttransgenic mice have impaired primordial germ cell development during embryogenesisand significantly lowered fertility in the adult state. [100]. Consistently, a deficiency inLin28A lowers the ATP/AMP ratio and NADH/NAD ratio in embryonic growth and hin-ders the rate of tissue repair [101]. Overall, Lin28 acts as a double sword that reprogramsglucose metabolism to induce the Warburg effect in cancers and acts as a factor necessaryfor healthy developmental processes.7. Lin28 Targets Various Messenger RNABesides microRNAs, Lin28 also targets messenger RNAs in a cell type-dependentand region-specific manner [102,103]. Similar to conventional mRNA binding proteins,Lin28A/B binds an array of similar RNA sequence motifs [103]. Those motifs are character-ized by uridine-rich fragments surrounded by guanosine residues. Lin28A/B increases theprotein levels of cell cycle regulators predominantly by stabilizing their respective mRNAsthrough interaction with the exonic regions and 3’ UTR of post-splicing mRNAs [103]. Theconserved GGAGA sequence shared between most of the let-7 members was also foundin many Lin28-targeted mRNAs [104]. By interacting with the RNA motifs in mRNAs,Lin28A/B serves as both positive and negative regulator of mRNA processing and transla-tion. This was reported to occur through interactions with protein factors such as hnRNP F,TIA-1, FUS/TLS, and TDP-43 [104]. Interestingly, Lin28A/B autoregulates its own mRNAfor translation [104]. These findings indicated that Lin28 has a broad range of functionsother than controlling let-7 miRNA expression.Moreover, Lin28 is a global regulator of different cellular compartments, includingstress granules (SGs) and cytoplasmic processing bodies (P-bodies) [105]. The formation ofSGs is a characteristic response of cancer cells when encountering therapy-induced stress.SGs contribute to cancer cell survival by reducing stress-related damage from oxidative,pH, and temperature fluctuations [105–107]. In SGs, Lin28A/B were found to bind T cellintracellular antigen 1 (TIA 1), an RNA-binding protein that stabilizes the mRNA andregulates splicing patterns [105,107]. Overexpression of TIA-1 induces SG assembly andalters gene expression and signal transduction patterns to promote cancer proliferation andmetastasis [106,108]. These studies indicate that Lin28 may play a key role in regulatingthe survival response of tumour cells when encountering therapy-induced stress.8. Lin28 and Prostate Cancer (PC)Prostate cancer (PC) is one of the most common malignancies in men worldwide [109].Even though PC generally has a relatively slow growth rate and low mortality rate, therapy-resistant CRPC poses a significant threat and takes thousands of lives every year [110].These tumours fail to respond to the first-line androgen deprivation therapies due to theloss of conventional dependency on the AR signalling for growth [111]. Many tumoursprogress into even more aggressive states by changing cancer cell lineage and transform intotherapy-induced neuroendocrine prostate cancers (t-NEPCs) that have a median survivalof less than one year [112]. Thus, there is a clear need for a comprehensive understandingof the biological mechanisms underlying PC development and its progression to developeffective treatments. Lin28 has been identified as one of the most promising candidates thatregulate therapy-resistant tumour progression [21]. Herein, we discuss the role of Lin28 inthe development and progression of PC.Cancers 2022, 14, 4640 13 of 23Early studies linked the role of Lin28 to the development of CRPC through the ob-servation that AR expression was regulated by the members of let-7 through c-Myc. Highlevels of Lin28A-suppressed let-7 in CRPC cells were found to indirectly stimulate AR ex-pression [113]. A follow-up study confirmed that elevated levels of let-7c in PC xenograftssignificantly reduced tumour growth and invasion [114]. Furthermore, PC patient tissueswere found to have a higher level of Lin28A protein than the benign tissues. Borrego-Diaz et al. [51] also reported that Lin28 is an important driver of PC, whereby Lin28B regu-lates c-Myc expression indirectly by binding to the intermediate c-Myc miRNA suppressormiR-212. Further mechanistic studies have shown that the LNCaP PC cell lines overexpress-ing Lin28A rendered these cells more resistant to antiandrogens, including enzalutamide,abiraterone, and bicalutamide [115]. The resistance was attributed to the Lin28-dependentstimulation of overexpression of the AR splice variant AR-V7. Lovnicki et al. [16] foundthat Lin28B, along with another core pluripotency stem cell gene, Sox2, are key drivers oft-NEPC [16]. Importantly, Lin28B induced a stem-like gene network and neuroendocrinebiomarkers, including HMGA2, that stimulated the transition of AdPC to more aggressivet-NEPC. Furthermore, Lin28B stimulated EMT properties, which promote t-NEPC forma-tion, by reducing N cadherin and increasing E cadherin. Similarly, another group illustratedthat upregulation of Sox2 via Lin28B induces androgen ablation-induced neuroendocrinedifferentiation via neuroendocrine (NE) lineage in prostate luminal cells [116]. Overall, theLin28/let-7 pathway regulates several downstream gene networks in CRPC progression.The expression of Lin28 is regulated by oncogenic pathways such as ESE3/EHF [117].In normal cells, ESE3/EHF binds Lin28 promoters and inhibits its transcription, whichresults in an upregulation of let-7 miRNA [118]. In this way, ESE3/EHF maintains a bal-ance between self-renewal and differentiation of normal prostatic epithelium. Significantdownregulation of ESE3/EHF was reported in PC [119,120]. It was estimated that approxi-mately 50% of PCs acquired ETS dysregulation through gene rearrangement [117]. Lin28silencing, both in vitro and in vivo, resulted in similar phenotypes in PC cells expressinghigh levels of ESE3/EHF. In particular, there was a decrease in PC cell spheroid formationand self-renewal abilities, indicating that Lin28 promotes tumourigenic and CSC-like phe-notypes [52]. In addition, the effects of Lin28 silencing were tested on NOD scid gamma(NSG) mice, where tumourigenic ESE3KD-PrEC cells transfected with siRNA against Lin28caused a reduction in xenograft growth in mice [52]. These findings suggest that ESE3/EHFis a key regulator of Lin28 expression in PCa.9. Therapeutic Inhibition of Lin28Despite a widespread view that RNA-binding proteins are undruggable, there is agrowing body of research on targeting Lin28A and Lin28B with small molecules [121–123].These small molecules bind to the surface of either CSD or ZKD of Lin28 and preventLin28/let-7 interaction from increasing let-7 biogenesis in cancer cells, resulting in sup-pression of cancer CSC and EMT phenotypes (Table 2). Notably, the studies outlined inthis section employed novel high-throughput screening techniques, including fluorescencepolarization (FP) and fluorescence resonance energy transfer (FRET), which are gainingpopularity in screenings for RNA-binding protein inhibitors [124,125]. These novel tech-nologies have enabled screenings of libraries containing up to 100,000 compounds (Table 2).The basic principle of the two most commonly used techniques, FP and FRET, involvesthe use of fluorescently labeled macromolecules that excite light differently upon RNA–protein interactions [126]. In this way, an event of inhibition between Lin28 and let-7 RNAcan be tracked.Cancers 2022, 14, 4640 14 of 23Table 2. Lin28 inhibitors. Lin28A and B expression in human cancer cell lines was confirmed fromthe Cancer Cell Line Encyclopedia (CCLC) Dependency Map database.TargetDomain Lin28 Inhibitor Identification Method Activities Ref.Coldshockdomain(CSD)LI71Cancers 2022, 14, x 15 of 25 Cold shock domain (CSD) LI71 FP-based HTS of 101,017 compounds from 17 libraries (1) Oligouridylation TUT IC50 = 27 μM (2) FP IC50 = 7 μM (3) Direct binding: NMR spectroscopy (4) Let-7 qPCR: 2–5 times (let-7b/c/d/f/g/i) in K562 (Lin28B+) at 100 μM 3–7 times (let-7a/b/c/d/e/f/g/i) in DKO+A (Lin28A+) mESCs at 100 μM [127] KCB3602 FRET-based HTS of 8400 compounds fromKorea Chemical Bank (KCB) (1) EMSA IC50 = 4.8 μM (2) Direct binding: SPR kD = 5.9 ± 2.3 μM (3) Let-7 qRT-PCR: 1.5–2.5 times (let-7a/b/d/f/g/i/miR-98) in JAR cells (Lin28A+ , Lin28B+) at 10 μM [128] Compound 1 FRET-based HTS of 4500 drug-like com-pounds (1) FRET IC50 = 4.03 μM (2) EMSA IC50 = 6.75–11.8 μM (Lin28A/B: let-7a-1/g) (3) Direct binding: SPR kD = 3.51 μM (4) Let-7 qPCR: ~1.5–2.5 times (let-7a/d/f/g/i/miR-98) in JAR cells (Lin28A+ , Lin28B+) at 40 μM ~1.5–2.3 times (let-7a/g) in PA-1 cells (Lin28A+ , Lin28B+) at 40 μM [129] C902 FP-based HTS of 15,000 natural prod-uct-inspired com-pounds (1) FP IC50 = 5.0 μM (2) Let-7 RT-qPCR: ~2 times (let-7a/g) in JAR cells (Lin28A+ , Lin28B+) at 20 μM [130] GG-43 Custom chemistry modifications of LI71 (1) EMSA IC50 = 21.9 μM (LI71 EMSA IC50 = 41.6 μM) (2) FP IC50 = 4 μM (LI71 FP IC50 = 7 μM) [131] Zinc finger do-main (ZKD) TPEN Fluorescence polari-zation (1) FP IC50 = 2.5 μM (2) Toxic in Lin28+ mESCs and Lin28- HeLa cells (Lin28B+) (3) Direct binding: NMR spectroscopy [127] FP-based HTS of 101,017compounds from 17li r ri(1) Oligouridylation TUT IC50 = 27 µM(2) FP IC50 = 7 µM(3) Direct binding: NMR spectroscopy(4) Let-7 qPCR:2–5 times (let-7b/c/d/f/g/i) in K562(Lin28B+) at 100 µM3–7 times (let-7a/b/c/d/e/f/g/i) inDKO+A (Lin28A+) mESCs at 100 µM[127]CB3602Cancers 2022, 14, x 15 of 25 Cold shock domain (CSD) LI71 FP-based HTS of 101,017 compounds from 17 libraries (1) Oligouridylation TUT IC50 = 27 μM (2) FP IC50 = 7 μM (3) Direct binding: NMR spectroscopy (4) Let-7 qPCR: 2–5 times (let-7b/c/d/f/g/i) in K562 (Lin28B+) at 100 μM 3–7 times (let-7a/b/c/d/e/f/g/i) in DKO+A (Lin28A+) mESCs at 100 μM [127] KCB3602 FRET-based HTS of 8400 compounds fromKorea Chemical Bank (KCB) (1) EMSA IC50 = 4.8 μM (2) Direct binding: SPR kD = 5.9 ± 2.3 μM (3) Let-7 qRT-PCR: 1.5–2.5 times (let-7a/b/d/f/g/i/miR-98) in JAR cells (Lin28A+ , Lin28B+) at 10 μM [128] Compound 1 FRET-based HTS of 4500 drug-like com-pounds (1) FRET IC50 = 4.03 μM (2) EMSA IC50 = 6.75–11.8 μM (Lin28A/B: let-7a-1/g) (3) Direct binding: SPR kD = 3.51 μM (4) Let-7 qPCR: ~1.5–2.5 times (let-7a/d/f/g/i/miR-98) in JAR cells (Lin28A+ , Lin28B+) at 40 μM ~1.5–2.3 times (let-7a/g) in PA-1 cells (Lin28A+ , Lin28B+) at 40 μM [129] C902 FP-based HTS of 15,000 natural prod-uct-inspired com-pounds (1) FP IC50 = 5.0 μM (2) Let-7 RT-qPCR: ~2 times (let-7a/g) in JAR cells (Lin28A+ , Lin28B+) at 20 μM [130] GG-43 Custom chemistry modifications of LI71 (1) EMSA IC50 = 21.9 μM (LI71 EMSA IC50 = 41.6 μM) (2) FP IC50 = 4 μM (LI71 FP IC50 = 7 μM) [131] Zinc finger do-main (ZKD) TPEN Fluorescence polari-zation (1) FP IC50 = 2.5 μM (2) Toxic in Lin28+ mESCs and Lin28- HeLa cells (Lin28B+) (3) Direct binding: NMR spectroscopy [127] FRET-base HTS of 8400compounds from KoreaChemical Bank (KCB)(1) EMSA IC50 = 4.8 µM(2) Direct binding: SPR kD = 5.9 ±2.3 µM(3) Let-7 qRT-PCR:1.5–2.5 times(let-7a/b/d/f/g/i miR-98) in JAR cells(Lin28A+, Lin28B+) at 10 µM[128]Co pound 1Cancers 2022, 14, x 15 of 25 Cold shock domain (CSD) LI71 FP-based HTS of 101,017 compounds from 17 libraries (1) Oligouridylation TUT IC50 = 27 μ (2) FP IC50 = 7 μM (3) Direct binding: NMR spectroscopy (4) Let-7 qPCR: 2–5 times (let-7b/c/d/f/g/i) in K562 (Lin28B+) at 100 μM 3–7 times (let-7a/b/c/d/e/f/g/i) in DKO+A (Lin28A+) mESCs at 100 μM [127] KCB3602 FRET-based HTS of 8400 comp unds fromKorea Chemical Bank(KCB) (1) EMSA IC50 = 4.8 μM (2) Direct binding: SPR kD = 5.9 ± 2.3 μM (3) Let-7 qRT-PCR: 1.5–2.5 times (let-7a/b/d/f/g/i/miR-98) in JA cells (Lin28A+ , i 28B+) at 10 μM [128] pound 1 FRET-based HTS of 4500 drug like com-pounds (1) FRET IC50 = 4.03 μM (2) EMSA IC50 = 6.75–11.8 μM (Lin28A/B: let-7a-1/g) (3) Direct binding: SPR kD = 3.51 μM (4) Le -7 qPCR: ~1.5–2.5 times (let-7a/d/f/g/i/miR-98) in JAR cells (Lin28A+ , Lin28B+) at 40 μM ~1.5–2.3 times (let-7a/g) in PA-1 cells (Lin28A+ , L n28B+) a 40 μM [129] C902 FP-based HTS of 15,000 natural prod-uct-inspired com-pounds (1) FP IC50 = 5.0 μM (2) Let-7 RT-qPCR: ~2 times (let-7a/g) in JAR cells (Lin28A+ , Lin28B+) at 20 μM [130] GG-43 Custom chemistry modifications of LI71 (1) EMSA IC50 = 21.9 μM (LI71 EMSA IC50 = 41.6 μM) (2) FP IC50 = 4 μM (LI71 FP IC50 = 7 μM) [131] Zinc finger do-main (ZKD) TPEN Fluorescence polari-zation (1) FP IC50 = 2.5 μM (2) Toxic in Lin28+ mESCs and Lin28- HeLa cells (Lin28B+) (3) Direct binding: NMR spectroscopy [127] FRET-based HTS of 4500drug-like compounds(1) FRET IC50 = 4.03 µM(2) EMSA IC50 = 6.75–11.8 µM(Lin28A/B: let-7a-1/g)(3) Direct binding: SPR kD = 3.51 µM(4) Let-7 qPCR:~1.5–2.5 times (let-7a/d/f/g/i/miR-98)in JAR cells (Lin28A+, Lin28B+) at40 µM~1.5–2.3 times (let-7a/g) in PA-1 cells(Lin28A+, Lin28B+) at 40 µM[129]C902Cancers 2022, 14, x 15 of 25 Cold shock domain (CSD) LI71 FP-based HTS of 101,017 compounds from 17 libraries (1) Oligouridylation TUT IC50 = 27 μM (2) FP IC50 = 7 μM (3) Direct binding: NMR spectroscopy (4) Let-7 qPCR: 2–5 times (l t-7 /c/d/f/g/i) in K562 (Lin28B+) at 100 μM 3–7 times (let-7a b c d/e/f g/i) in DKO+A (Lin28A+) mESCs at 100 μM [127] KCB3602 FRET-based HTS of 8400 compounds fromKorea Chemical Bank(KCB) (1) EMSA IC50 = 4.8 μM (2) Direct binding: SPR kD = 5.9 ± 2.3 μM (3) Le -7 qRT-PCR: 1.5–2.5 times (let-7a/b/d/f/g/i/miR-98) in JAR cells (Lin28A+ , Lin28B+) at 10 μM [128] Compound 1 FRET-based HTS of 4500 drug-like com-poun s (1) FRET IC50 = 4.03 μM (2) EMSA IC50 = 6.75–11.8 μM (Lin28A/B: let-7a-1/g) (3) Direct binding: SPR kD = 3.51 μM (4) Let-7 qPCR: ~1.5–2.5 t m s (let-7a/d/f/g/i/miR-98) in JAR cells (Lin28A+ , Lin28B+) at 40 μM ~1.5–2.3 times (let-7a/g) in PA-1 cells (L 28A+ , Lin28B+) at 40 μM [129] C902 FP-based HTS of 15,000 natural prod-uct-inspired com-pounds (1) FP IC50 = 5.0 μM (2) Let-7 T-qPCR: ~2 times (let-7a/g) in JAR cells (Lin28A+ , Lin28B+) at 20 μM [130] GG-43 Custom chemistry modifications of LI71 (1) EMSA IC50 = 21.9 μM (LI71 EMSA IC50 = 41.6 μM) (2) FP IC50 = 4 μM (LI71 FP IC50 = 7 μM) [131] Zinc finger do-main (ZKD) TPEN Fluorescence polari-zation (1) FP IC50 = 2.5 μM (2) Toxic i Lin28+ mESCs and Lin28- HeLa cells (Lin28B+) (3) Direct binding: NMR spectroscopy [127] FP-b sed HTS of 15,000atural product-inspiredcompounds(1) FP IC50 = 5.0 µM(2) Let-7 RT-qPCR:~2 times (let-7a/g) in JAR cells( i 28A+, Lin28B+) at 20 µM[130]GG-43Cancers 2022, 14, x 15 of 25 Cold shock domain (CSD) LI71 FP-based HTS of 101,017 co pounds fro 17 libraries (1) Oligouridylation TUT IC50 = 27 μ (2) FP IC50 = 7 μ (3) Direct binding: N R spectroscopy (4) Let-7 qPCR: 2–5 ti es (let-7b/c/d/f/g/i) in K562 (Lin28B+) at 100 μ 3–7 ti es (let-7a/b/c/d/e/f/g/i) in DKO+A (Lin28A+) ESCs at 100 μ [127] KCB3602 FRET-based HTS of 8400 co pounds froKorea Che ical Bank (KCB) (1) E SA IC50 = 4.8 μ (2) Direct binding: SPR kD = 5.9 ± 2.3 μ (3) Let-7 qRT-PCR: 1.5–2.5 ti es (let-7a/b/d/f/g/i/ iR-98) in JAR cells (Lin28A+ , Lin28B+) at 10 μ [128] Co pound 1 FRET-based HTS of 4500 drug-like co -poun s (1) FRET IC50 = 4.03 μ (2) E SA IC50 = 6.75–11.8 μ (Lin28A/B: let-7a-1/g) (3) Direct binding: SPR kD = 3.51 μ (4) Let 7 qPCR: ~1.5–2.5 ti es (let-7a/d/f/g/i/ iR-98) in JAR cells (Lin28A+ , Lin2 B+) at 40 μ ~1.5–2.3 ti es (let-7a/g) in PA-1 cells (Li 28A+ , Lin28B+) at 40 μ [129] C902 FP-based HTS of 15,000 n tu al prod-uct inspired co -pounds (1) FP IC50 = 5.0 μ (2) Let-7 RT-qPCR: ~2 ti es (let-7a/g) in JAR cells (Lin28A+ , Lin B ) at 20 μ [130] GG-43 Cust che istry odifications of LI71 (1) E SA IC50 = 21.9 μ (LI71 E SA IC50 = 41.6 μ ) (2) FP IC50 = 4 μ (LI71 FP IC50 = 7 μ ) [131] Zinc finger do-main (ZKD) TPEN Fluorescence polari-zation (1) FP IC50 = 2.5 μ (2) Toxic in Lin28+ ESCs and Lin28- HeLa cells (Lin28B+) (3) Direct binding: N R spectroscopy[127] Custom chemistrymodifications of LI71(1) EMSA IC50 = 21.9 µM (LI7 EMSAIC50 = 41.6 µM)(2) FP IC50 = 4 µM (LI71 FPIC50 = 7 µM)[131]Zincfingerdomain(ZKD) TPENCancers 2022, 14, x 15 of 25 Cold shock domain (CSD) LI71 FP-based HTS of 101,017 compounds from 17 libraries (1) Oligouridylation TUT IC50 = 27 μM (2) FP IC50 = 7 μM (3) Direct binding: NMR spectroscopy (4) Let-7 qPCR: 2–5 times (let-7b/c/d/f/g/i) in K562 (Lin28B+) at 100 μM 3–7 tim s (let-7a/b/c/d/e/f/g/i) in DKO+A (Lin28A+) mESCs at 100 μM [127] KCB3602 FRET-based HTS of 8400 compounds fromKorea Chemical Bank (KCB) (1) EMSA IC50 = 4.8 μ (2) Direct binding: SPR kD = 5.9 ± 2.3 μM (3) Let-7 qRT-PCR: 1.5–2.5 times (let-7a/b/d/f/g/i/miR-98) in JAR cells (Lin28A+ , Lin28B+) at 10 μM [128] Compound 1 FRET-based HTS of 4500 drug-like com-pounds (1) FRET IC50 = 4.03 μM (2) EMSA IC50 = 6.75–11.8 μM (Lin28A/B: let-7a-1/g) (3) Direct binding: SPR kD = 3.51 μM (4) Let-7 qPCR: ~1.5–2.5 times (let-7a/d/f/g/i/mi -98) in JAR cells (Lin28A+ , Lin28B+) at 40 μM 3 g) in PA-1 cells (Lin28A+ , Lin28B+) at 40 μM [129] C902 FP-based HTS of 15,000 natural prod-uct-inspired c m-pounds (1) FP IC50 = 5.0 μM (2) Let-7 RT-qPCR: ~2 times (let-7a/g) in JA cells (Lin28A+ , Lin28B+) at 20 μM [130] G-43 Custom chemistry modifications of LI71 (1) EMSA IC50 = 21.9 μM (LI71 EMSA IC50 = 41.6 μM) (2) FP I = 4 μM (LI71 FP IC50 = 7 μM) [131] Zinc finger do-main (ZKD) TPEN Fluorescence polari-zation (1) FP IC50 = 2.5 μM (2) Toxic in Lin28+ mESCs and Lin28- HeLa cells (Lin28B+) (3) Direct binding: NMR spectroscopy [127] Fluorescence polarization(1) FP IC50 = 2.5 µM(2) Toxic in Lin28+mESCs and - HeLa cells (Lin28B+)(3) Direct bi ding: NMR spectroscopy[127]Nb-S2A4Functional EpitopeNanobody SelectionPlatform(1) Oligouridylation TUT IC50 = 0.2 µM(2) Let-7 qPCR:3–12 times(let-7b/c/d/e/f/g/i/miR-20b) in HeLacells (Lin28B+)[132]Cancers 2022, 14, 4640 15 of 23Table 2. Cont.TargetDomain Lin28 Inhibitor Identification Method Activities Ref.UnknownSB/ZW/0065Cancers 2022, 14, x 16 of 25 Nb-S2A4 Functional Epitope Nanobody Selection Platform (1) Oligouridylation TUT IC50 = 0.2 μM (2) Let-7 qPCR: 3–12 times (let-7b/c/d/e/f/g/i/miR-20b) in HeLa cells (Lin28B+) [132] Unknown SB/ZW/0065 FP-based HTS of 2,768 compounds from Sigma LOPAC1280 library, NCI diversity set II, and a targeted nucleic acid structure library (1) FP IC50 = 7.05± 0.13 μM [133] 6-hydroxy-DL-DOPA (1) FP IC50 = 4.71 ± 0.16 μM CCG-234459 Cat-ELCCA-based HTS of 127,007 compounds from LOPAC, Prest-wick, Maybridge, ChemDiv, and UMich libraries (1) Cat-ELCCA IC50 = 8.3 μM [134] CCG-233094 (1) Cat-ELCCA IC50= 10.3 μM 1632 FRET-based HTS of 16,000 compounds from Maybridge Hit-finder library (1) ELISA IC50= 8 μM (2) On target: SPR (3) Clonogenic assay: GI50 = 20–80 μM (4) Tumour sphere-formation assay: GI50 = ~26 μM (5) Let-7 RT-qPCR: 1.2–3 times (let-7a/f/g) in Huh7 cells (Lin28A+ , Lin28B+) at 60 μM 1.4–2 times (let-7a/g) in JAR cells (Lin28A+ , Lin28B+) at 20 μM [97,135,136] FP-based HTS of 2,768compounds from SigmaLOPAC1280 library, NCIdiversity set II, and atargeted nucleic acidst ucture library(1) FP IC50 = 7.05± 0.13 µM[133]6-hydroxy-DL-DOPACancers 2022, 14, x 16 of 25 Nb-S2A4 Functional Epitope Nanobody Selection Platform (1) Oligouridylation TUT IC50 = 0.2 μM (2) Let-7 qPCR: 3–12 times (let-7b/c/d/e/f/g/i/miR-20b) in HeLa cells (Lin28B+) [132] Unknown SB/ZW/0065 FP-based HTS of 2,768 compounds from Sigma LOPAC1280 library, NCI diversity set II, and a targeted nucleic acid st ct re library (1) FP IC50 = 7.05± 0.13 μM [133] 6-hydroxy-DL-DOPA (1) FP IC50 = 4.71 ± 0.16 μM CCG-234459 Cat-ELCCA-based HTS of 127,007 compounds from LOPAC, Pre t-wick, Maybridge, ChemDiv, and UMich libraries (1) Cat-ELCCA IC50 = 8.3 μM [134] CCG-233094 (1) Cat-ELCCA IC50= 10.3 μM 1632 FRET-based HTS of 16,000 compounds from Maybridge Hit-finder library (1) ELISA IC50= 8 μM (2) On target: SPR (3) Clonogenic assay: GI50 = 20–80 μM (4) Tumour sphere-formation assay: GI50 = ~26 μM (5) Let-7 RT-qPCR: 1.2–3 times (let-7a/f/g) in Huh7 cells (Lin28A+ , Lin28B+) at 60 μM 1.4–2 times (let-7a/g) in JAR cells (Lin28A+ , Lin28B+) at 20 μM [97,135,136] (1) FP IC50 = 4.71 ± 0.16 µMCCG-234459Cancers 2022, 14, x 16 of 25 Nb-S2A4 Functional Epitope Nanobody Selection Platform (1) Oligouridylation TUT IC50 = 0.2 μM (2) Let-7 qPCR: 3–12 times (let-7b/c/d/e/f/g/i/miR-20b) in HeLa cells (Lin28B+) [132] Unknown SB/ZW/0065 FP-based HTS of 2,768 compounds from Sigma LOPAC1280 library, NCI diversity set II, and a ta geted nucleic acid structure library (1) FP IC50 = 7.05± 0.13 μM [133] 6-hydroxy-DL-DOPA (1) FP IC50 = 4.71 ± 0.16 μM CCG-234459 Cat-ELCCA-based HTS of 127,007 compounds from LOPAC, Prest-wick, Maybridge, ChemDiv, and UMich libraries (1) Cat-ELCCA IC50 = 8.3 μM [134] CCG-233094 (1) Cat-ELCCA IC50= 10.3 μM 1632 FRET-based HTS of 16,000 compounds from Maybridge Hit-finder library (1) ELISA IC50= 8 μM (2) On target: SPR (3) Clonogenic assay: GI50 = 20–80 μM (4) Tumour sphere-formation assay: GI50 = ~26 μM (5) Let-7 RT-qPCR: 1.2–3 times (let-7a/f/g) in Huh7 cells (Lin28A+ , Lin28B+) at 60 μM 1.4–2 times (let-7a/g) in JAR cells (Lin28A+ , Lin28B+) at 20 μM [97,135,136] Cat-ELCCA-based HTSof 12 ,007 compoundsfr , stwick,Maybridge, ChemDiv,and UMich libraries(1) Cat-ELCCA IC50 = 8.3 µM[134]CCG-233094Cancers 2022, 14, x 16 of 25 Nb-S2A4 Functional Epitope Nanobody Selection Platform (1) Oligouridylation TUT IC50 = 0.2 μM (2) Let-7 qPCR: 3–12 times (let-7b/c/d/e/f/g/i/miR-20b) in HeLa cells (Lin28B+) [132] Unknown SB/ZW/0065 FP-based HTS of 2,768 compounds from Sigma LOPAC1280 library, NCI diversity set II, and a targeted nucleic acid structure library (1) FP IC50 = 7.05± 0.13 μM [133] 6-hydroxy-DL-DOPA (1) FP IC50 = 4.71 ± 0.16 μM CCG-234459 Cat-ELCCA-based HTS of 127,007 compounds from LOPAC, Prest-wick, Maybridge, Che Div, and UMich libraries (1) Cat-ELCCA IC50 = 8.3 μM [134] CCG-233094 (1) Cat-ELCCA IC50= 10.3 μM 1632 FRET-based HTS of 16,000 compounds from Maybridge Hit-finder library (1) ELISA IC50= 8 μM (2) On target: SPR (3) Clonogenic assay: GI50 = 20–80 μM (4) Tumour sphere-formation assay: GI50 = ~26 μM (5) Let-7 RT-qPCR: 1.2–3 times (let-7a/f/g) in Huh7 cells (Lin28A+ , Lin28B+) at 60 μM 1.4–2 times (let-7a/g) in JAR cells (Lin28A+ , in28 +) at 20 [97,135,136] (1) Cat-ELCCA IC50 = 10.3 µM1632Cancers 2022, 14, x 16 of 25 Nb-S2A4 Functional Epitope Nanobody Selection Platform (1) Oligouridylation TUT IC50 = 0.2 μM (2) Let-7 qPCR: 3–12 times (let-7b/c/d/e/f/g/i/miR-20b) in HeLa cells (Lin28B+) [132] Unknown SB/ZW/0065 FP-based HTS of 2,768 compounds from Sigma LOPAC1280 library, NCI diversity set II, and a targeted nucleic acid structure library (1) FP IC50 = 7.05± 0.13 μM [133] 6-hydroxy-DL-DOPA (1) FP IC50 = 4.71 ± 0.16 μM CCG-234459 Cat-ELCCA-based HTS of 127,007 compounds from LOPAC, Prest-wick, Maybridge, Che Div, and UMich libraries (1) Cat-ELCCA IC50 = 8.3 μM [134] CCG-233094 (1) Cat-ELCCA IC50= 10.3 μM 1632 FRET-based HTS of 16,000 compounds from Maybridge Hit-finder library (1) ELISA IC50= 8 μM (2) On target: SPR (3) Clonogenic assay: GI50 = 20–80 μM (4) Tumour sphere-formation assay: GI50 = ~26 μM (5) Let-7 RT-qPCR: 1.2–3 times (let-7a/f/g) in Huh7 cells (Lin28A+ , Lin28B+) at 60 μM 1.4–2 times (let-7a/g) in JAR cells (Lin28A+ , Lin28B+) at 20 μM [97,135,136] FRET-based HTS of16,000 compounds fromMaybridge Hitfinderlibrary(1) ELISA IC50 = 8 µM(2) On target: SPR(3) Clonogenic assay GI50 = 20–80 µM(4) Tumour sphere-formation assay:GI50 = ~26 µM(5) Let-7 RT-qPCR:1.2–3 tim s (let-7a/f/g) in Huh7 cells(Lin28A+, Lin28B+) at 60 µM1.4–2 times (let-7a/g) in JAR cells(Lin28A+, Lin28B+) at 20 µM[97,135,136]KCB170522Cancers 2022, 14, x 17 of 25 KCB170522 Fluorescence inten-sity-based (FL)-based HTS of Korea Chemi-cal Bank (KCB), natu-ral products, and drug-like libraries (1) FL IC50 = 9.55 (2) EMSA IC50= 12.8 μM (3) Let-7 RT-qPCR: - 1.4–2 times (let-7a/g) in JAR cells (Lin28A+ , Lin28B+) at 20 μM [137] 9.1. Lin28 Inhibitor Screening: Fluorescence Resonance Energy Transfer The earliest implementation of the FRET assay for Lin28 inhibitor screening yielded compound 1632, which successfully restored let-7 levels in Huh7 cells and reduced “stem-ness” in mouse embryonic stem cells (mESC) [136]. Compound 1632 suppressed mRNA levels of known markers of pluripotency, including Pou5f1/Oct4, Rex1, and Stella, and increased differentiation. Furthermore, the authors demonstrated that 1632 effectively in-hibited clonogenic growth in 22Rv1, PC3, DU145, and Huh7 cell lines and induced dose-dependent reduction of tumour-sphere formation in 22Rv1 and Huh7 cells. Subsequent studies demonstrated that 1632 downregulates Lin28 mRNA levels, increases let-7 miRNA levels, and, importantly, suppresses cell surface expression of PD-L1, one of the key proteins involved in immune evasion of cancer cells [135]. 1632 was reported to block glycolytic, tumour sphere-forming, and migratory capacities in MDA-MB-231 and H1299 cell lines, as well as reduce the stemness phenotype [97]. The pharmacological effects of 1632 are also observed in vivo, as it inhibited tumour growth and metastasis in immuno-deficient orthotopic mouse models of MDA-MB-231 human breast cancer cells. Addition-ally, compound 1632 suppresses cell growth and clonogenicity in acute myeloid leukemia cell lines and tissue samples [62]. The mechanisms involved in the inhibition of the NFkB pathway induced leukemic stem cells and cell cycle arrest at the G1/S phase. Collectively, these studies demonstrated that compound 1632 is a promising drug candidate for Lin28 overexpressing tumours. Other compounds identified by FRET also showed some promising activity in vitro. In particular, compound KCB3602 was found to increase let-7 levels in human choriocar-cinoma JAR cells and, subsequently, downregulate gene products of the key drivers of CSC, including HMGA, IGF2, IMR1, Ras, and Lin28A [128]. Furthermore, KCB3602 effec-tively suppressed the sphere-like growth of cancer cells, a hallmark of CSCs. The authors confirmed the on-target effects using surface plasmon resonance (SPR) assays and showed that KCB3602 does not impair cell growth in a non-cancerous cell line. Another small mol-ecule, benzopyranylpyrazole-based compound (1), was found to upregulate let-7 levels in JAR and PA-1 cell lines [129]. It also downregulated the expression of let-7 target genes, including HMGA2, c-Myc, and Ras. These compounds proved that the pharmacological inhibition of Lin28 suppresses CSC gene signatures. However, further investigations are necessary to determine if these inhibitory effects can be advanced to in vivo studies. 9.2. Lin28 Inhibitor Screening: Fluorescence Polarization Another commonly used technique is the FP assay, which was first used to screen for Lin28 inhibitors by Lightfoot and colleagues in 2016 [133]. The authors reported two small molecule inhibitors, SB/ZW/0065 and 6-hydroxy-DL-DOPA, had low micromolar activity in FP assays. Electrophoresis mobility shift assay (EMSA) was then used to confirm the disruption of Lin28/pre-let-7g complex. Furthermore, these molecules were shown to par-tially restore Dicer cleavage activity of pre-let-7g in vitro but failed to restore let-7g levels in P19 embryonal carcinoma cells. Later, Wang et al. [133] also employed FP screening of a large library and presented two small molecule inhibitors—namely, LI71 and TPEN—that targeted the CSD and ZKD domains of Lin28A and Lin28B, respectively. Importantly, Fluorescenceintensity-based(FL)-based HTS of KoreaChemical ank (KCB),natural products, anddrug-like libraries(1) FL IC50 = 9.55(2) EMSAIC50 = 12.8 µM(3) Let-7 RT-qPCR:- 1.4–2 times (let-7a/g) in JAR cells(Lin28A+, Lin28B+) at 20 µM[137]9.1. Lin28 Inhibitor Screening: Fluorescence Resonance Energy TransferThe earliest implementation of the FRET assay for Lin28 inhibitor screening yieldedcompound 1632, which successfully restored let-7 levels in Huh7 cells and reduced “stem-ness” in mouse e bryonic stem cells (mESC) [136]. Compound 1632 suppresse mRNAlevels of known m rkers of pluripotency, inclu ing Pou5f1/Oct4, R x1, and Stella, andincreased diff rentiation. Furthermore, the authors demonstrate that 1632 effectivelyinhibited clonogenic growth in 22Rv1, PC3, DU145, and cell lines and induceddose-dependent reduction of tumour-sphere formation in 22Rv1 and Huh7 cells. Subse-Cancers 2022, 14, 4640 16 of 23quent studies demonstrated that 1632 downregulates Lin28 mRNA levels, increases let-7miRNA levels, and, importantly, suppresses cell surface expression of PD-L1, one of thekey proteins involved in immune evasion of cancer cells [135]. 1632 was reported to blockglycolytic, tumour sphere-forming, and migratory capacities in MDA-MB-231 and H1299cell lines, as well as reduce the stemness phenotype [97]. The pharmacological effects of1632 are also observed in vivo, as it inhibited tumour growth and metastasis in immunode-ficient orthotopic mouse models of MDA-MB-231 human breast cancer cells. Additionally,compound 1632 suppresses cell growth and clonogenicity in acute myeloid leukemia celllines and tissue samples [62]. The mechanisms involved in the inhibition of the NFkBpathway induced leukemic stem cells and cell cycle arrest at the G1/S phase. Collectively,these studies demonstrated that compound 1632 is a promising drug candidate for Lin28overexpressing tumours.Other compounds identified by FRET also showed some promising activity in vitro.In particular, compound KCB3602 was found to increase let-7 levels in human choriocarci-noma JAR cells and, subsequently, downregulate gene products of the key drivers of CSC,including HMGA, IGF2, IMR1, Ras, and Lin28A [128]. Furthermore, KCB3602 effectivelysuppressed the sphere-like growth of cancer cells, a hallmark of CSCs. The authors con-firmed the on-target effects using surface plasmon resonance (SPR) assays and showedthat KCB3602 does not impair cell growth in a non-cancerous cell line. Another smallmolecule, benzopyranylpyrazole-based compound (1), was found to upregulate let-7 levelsin JAR and PA-1 cell lines [129]. It also downregulated the expression of let-7 target genes,including HMGA2, c-Myc, and Ras. These compounds proved that the pharmacologicalinhibition of Lin28 suppresses CSC gene signatures. However, further investigations arenecessary to determine if these inhibitory effects can be advanced to in vivo studies.9.2. Lin28 Inhibitor Screening: Fluorescence PolarizationAnother commonly used technique is the FP assay, which was first used to screenfor Lin28 inhibitors by Lightfoot and colleagues in 2016 [133]. The authors reported twosmall molecule inhibitors, SB/ZW/0065 and 6-hydroxy-DL-DOPA, had low micromolaractivity in FP assays. Electrophoresis mobility shift assay (EMSA) was then used to confirmthe disruption of Lin28/pre-let-7g complex. Furthermore, these molecules were shownto partially restore Dicer cleavage activity of pre-let-7g in vitro but failed to restore let-7g levels in P19 embryonal carcinoma cells. Later, Wang et al. [133] also employed FPscreening of a large library and presented two small molecule inhibitors—namely, LI71and TPEN—that targeted the CSD and ZKD domains of Lin28A and Lin28B, respectively.Importantly, the authors confirmed the direct binding of these molecules to their respectivetarget domains—i.e., CSD and ZKD—with protein nuclear magnetic resonance (NMR)spectroscopy. LI71 treatment upregulated various members of the let-7 family in leukemiacell line K562 and mouse embryonic stem cells, while TPEN was generally toxic in vitro.Using LI71 as a scaffold, further modifications resulted in derivatives with stronger potencyin FP and EMSA assays [131]. They performed structure–activity relationship (SAR) anddocking-based binding mode analyses and proposed that the binding mode features arenecessary to inhibit protein–RNA interaction at the Lin28 CSD interface. These featuresincluded a pi–cation interaction with Lys102 and several hydrogen bonds with Lys78, Lys98,and Ala101. Interestingly, another study performed a similar series of experiments (i.e.,FP HTS, SAR, docking) and presented a new class of trisubstituted pyrrolinone inhibitors,including the hit compound C902. This work highlighted the importance of Lys102 for thesuccessful inhibition of Lin28 CSD [130].9.3. Lin28 Inhibitor Screening: Other MethodsA few other studies have focused on the development of novel experimental high-throughput techniques in the context of Lin28/let-7 interaction inhibition. For instance,catalytic enzyme-linked click chemistry assay (cat-ELCCA) enabled the identification of twoN,N′-(1,2-phenylene)-dibenzenesulfonamide derivatives, CCG-233094 and CCG-234459 [134].Cancers 2022, 14, 4640 17 of 23The compounds were shown to inhibit Dicer-mediated cleavage of pre-let-7d and on-target effects confirmed by SPR. Another study focused on the development of a flu-orescence intensity-based (FL) assay to screen for potential Lin28/pre-let-7 interactiondisruptors [137]. The authors validated their method by identifying a novel scaffold,KCB170522, which was also active in EMSA and showed an expected increase in cellular let-7 levels. Besides small molecules, an epitope inhibitor named Nb-S2A4 was developed andshown to indirectly impact ZKD through recognition of N-terminal TUT4 and disruptionof its association with Lin28B ZKD/pre-let-7 complex [132]. Nb-S2A4 prevents pre-let-7degradation by allowing oligouridylation and monouridylation activities of TUT4 withoutstopping the formation of the Lin28B/pre-let-7 complex. In summary, several bioassayshave been established to screen and validate Lin28 inhibitors that can block Lin28/let-7interactions and suppress the CSC and EMT phenotypes of cancer cells. Although severalLin28 compounds had been discovered and developed, none have reached clinical trials,and more efforts in the field of research are needed.10. ConclusionsBoth Lin28A and Lin28B play important roles in cancer development and progression.As CSC promoters, they induce the stemness phenotype and drive the self-renewal andclonogenic capabilities of cancer cells. The Lin28-mediated stemness gene network conferstherapy resistance in various cell and xenograft models. It promotes cancer cell migrationand invasion, leading to cancer progression to more aggressive stages. Importantly, Lin28overexpression is observed in a wide range of cancer types, highlighting that it could beused as a common prognostic marker and therapeutic target of cancer.The development of Lin28 inhibitors as therapies is still at its early stage, as moststudies focus on the screening techniques and aim to provide proof-of-concept for Lin28inhibition. However, a few promising molecules have emerged and been shown to success-fully restore let-7 levels and downregulate downstream oncogenes. Furthermore, somestudies showed that the pharmacological effects of Lin28 modulation result in the sup-pression of tumour growth in animal models. Thus, the development of Lin28-targeteddrugs presents an attractive strategy for cancer treatment, especially in highly aggressivemetastatic forms. The rise of computational drug discovery should aid the development ofsuch drugs. The largest performed screening for Lin28 inhibitors covered around 100,000compounds, whereas the available chemical space counts in the billions nowadays. Thus,virtual screening coupled with novel in vitro screening techniques should make it possibleto explore the vast chemical space and find safe and efficacious small molecule inhibitorsof Lin28 protein.Author Contributions: Conceptualization, Z.L., M.R., and X.D.; writing—original draft preparation,Z.L.; Tables and figures—preparation, Z.L.; Tables and figures — review and editing, Z.L., M.R., andX.D.; writing—review and editing, Z.L., M.R., and X.D; supervision, X.D., A.C.; project administration,Z.L., M.R., A.C., and X.D. All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by DoD grant PC190327 and CIHR grants PJT-156150 and PJT-PJT-178063 to X.D.Conflicts of Interest: The authors declare no conflict of interest.References1. Dagogo-Jack, I.; Shaw, A.T. Tumour Heterogeneity and Resistance to Cancer Therapies. Nat. Rev. Clin. Oncol. 2018, 15, 81–94.[CrossRef]2. 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Lin28
let-7
metastasis
metabolism
epithelial-to-mesenchymal transition
prostate cancer
cancer stem cell
Lin28 inhibitors
Lin28 Regulates Cancer Cell Stemness for Tumour Progression
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