Tribbles 2 pseudokinase confers enzalutamide resistance in prostate cancer by promoting lineage plasticity

Enzalutamide, a second-generation antiandrogen, is commonly prescribed for the therapy of advanced prostate cancer, but enzalutamide-resistant, lethal, or incurable disease invariably develops. To understand the molecular mechanism(s) behind enzalutamide resistance, here, we comprehensively analyzed a range of prostate tumors and clinically relevant models by gene expression array, immunohistochemistry, and Western blot, which revealed that enzalutamide-resistant prostate cancer cells and tumors overexpress the pseudokinase, Tribbles 2 (TRIB2). Inhibition of TRIB2 decreases the viability of enzalutamide-resistant prostate cancer cells, suggesting a critical role of TRIB2 in these cells. Moreover, the overexpression of TRIB2 confers resistance in prostate cancer cells to clinically relevant doses of enzalutamide, and this resistance is lost upon inhibition of TRIB2. Interestingly, we found that TRIB2 downregulates the luminal markers androgen receptor and cytokeratin 8 in prostate cancer cells but upregulates the neuronal transcription factor BRN2 (Brain-2) and the stemness factor SOX2 (SRY-box 2) to induce neuroendocrine characteristics. Finally, we show that inhibition of either TRIB2 or its downstream targets, BRN2 or SOX2, resensitizes resistant prostate cancer cells to enzalutamide. Thus, TRIB2 emerges as a potential new regulator of transdifferentiation that confers enzalutamide resistance in prostate cancer cells via a mechanism involving increased cellular plasticity and lineage switching. Enzalutamide, a second-generation antiandrogen, is commonly prescribed for the therapy of advanced prostate cancer, but enzalutamide-resistant, lethal, or incurable disease invariably develops. To understand the molecular mechanism(s) behind enzalutamide resistance, here, we comprehensively analyzed a range of prostate tumors and clinically relevant models by gene expression array, immunohistochemistry, and Western blot, which revealed that enzalutamide-resistant prostate cancer cells and tumors overexpress the pseudokinase, Tribbles 2 (TRIB2). Inhibition of TRIB2 decreases the viability of enzalutamide-resistant prostate cancer cells, suggesting a critical role of TRIB2 in these cells. Moreover, the overexpression of TRIB2 confers resistance in prostate cancer cells to clinically relevant doses of enzalutamide, and this resistance is lost upon inhibition of TRIB2. Interestingly, we found that TRIB2 downregulates the luminal markers androgen receptor and cytokeratin 8 in prostate cancer cells but upregulates the neuronal transcription factor BRN2 (Brain-2) and the stemness factor SOX2 (SRY-box 2) to induce neuroendocrine characteristics. Finally, we show that inhibition of either TRIB2 or its downstream targets, BRN2 or SOX2, resensitizes resistant prostate cancer cells to enzalutamide. Thus, TRIB2 emerges as a potential new regulator of transdifferentiation that confers enzalutamide resistance in prostate cancer cells via a mechanism involving increased cellular plasticity and lineage switching. Enzalutamide, an inhibitor of androgen receptor (AR) function, is a popular drug commonly prescribed to treat advanced prostate cancer, but resistant prostate cancer eventually develops which grow aggressively, leading to widespread metastatic disease and ends up with a lethal outcome (1Armstrong A.J. Szmulewitz R.Z. Petrylak D.P. Holzbeierlein J. Villers A. Azad A. Alcaraz A. Alekseev B. Iguchi T. Shore N.D. Rosbrook B. Sugg J. Baron B. Chen L. Stenzl A. Arches: A randomized, phase III study of androgen deprivation therapy with enzalutamide or placebo in men with metastatic hormone-sensitive prostate cancer.J. Clin. Oncol. 2019; 37: 2974-2986Google Scholar, 2Kregel S. Chen J.L. Tom W. Krishnan V. Kach J. Brechka H. Fessenden T.B. Isikbay M. Paner G.P. Szmulewitz R.Z. Vander Griend D.J. Acquired resistance to the second-generation androgen receptor antagonist enzalutamide in castration-resistant prostate cancer.Oncotarget. 2016; 7: 26259-26274Google Scholar, 3Tucci M. Zichi C. Buttigliero C. Vignani F. Scagliotti G.V. Di Maio M. Enzalutamide-resistant castration-resistant prostate cancer: Challenges and solutions.Onco. Targets Ther. 2018; 11: 7353-7368Google Scholar). Based on present assessment, the enzalutamide resistant type of aggressive prostate cancer is responsible for most of the morbidity and mortality associated with prostate cancer and ∼30,000 lives of American men are lost every year (4Miller K.D. Fidler-Benaoudia M. Keegan T.H. Hipp H.S. Jemal A. Siegel R.L. Cancer statistics for adolescents and young adults, 2020.CA Cancer J. Clin. 2020; 70: 443-459Google Scholar). Nevertheless, the lack of proper understanding about critical molecular targets in hormone refractory, enzalutamide-resistant prostate cancer cells, largely contributes to majority of the prostate cancer fatalities. Clinical manifestation of drug resistance is the result of selective growth of cell clones that are either intrinsically capable of or have acquired the power to resist drug’s effects on critical survival mechanisms. However, knowledge about signaling mechanisms that play active roles in the progression phase of prostate cancer to enzalutamide resistance is still limited, which is a delaying progress toward the development of a long-term, effective therapeutic strategy. Common forms of prostate cancer cells bear luminal characteristics and depend on androgenic signaling for survival and growth, which is the basis for antiandrogenic therapy. However, it has been realized that men with prostate cancer who were treated with antiandrogenic therapies frequently develop aggressive and deadly forms of prostate cancers, which are no longer responsive to androgen-blockade therapies. Several reports encompassing the involvement of both AR reactivation or bypass, as well as androgen-independent signaling, have been forwarded to explain the mechanism of enzalutamide resistance. However, analysis of multiple cell lines and in vivo models, which were used to explore the molecular basis, have ended up with identification of cancer cell subtypes (5Blatt E.B. Raj G.V. Molecular mechanisms of enzalutamide resistance in prostate cancer.Cancer Drug Resist. 2019; 2: 189-197Google Scholar, 6Chen W.S. Aggarwal R. Zhang L. Zhao S.G. Thomas G.V. Beer T.M. Quigley D.A. Foye A. Playdle D. Huang J. Lloyd P. Lu E. Sun D. Guan X. Rettig M. et al.Genomic drivers of poor prognosis and enzalutamide resistance in metastatic castration-resistant prostate cancer.Eur. Urol. 2019; 76: 562-571Google Scholar). Current molecular understanding suggests that in addition to AR reactivation by mutation or splice variants, manifestation of enzalutamide resistance can be the result of overgrowth of cells that are developed in the tumor by lineage switching which may be triggered by drug-induced repression or loss of the AR-signaling (7Akamatsu S. Inoue T. Ogawa O. Gleave M.E. Clinical and molecular features of treatment-related neuroendocrine prostate cancer.Int. J. Urol. 2018; 25: 345-351Google Scholar, 8Beltran H. Prandi D. Mosquera J.M. Benelli M. Puca L. Cyrta J. Marotz C. Giannopoulou E. Chakravarthi B.V. Varambally S. Tomlins S.A. Nanus D.M. Tagawa S.T. Van Allen E.M. Elemento O. et al.Divergent clonal evolution of castration-resistant neuroendocrine prostate cancer.Nat. Med. 2016; 22: 298-305Google Scholar, 9Labrecque M.P. Coleman I.M. Brown L.G. True L.D. Kollath L. Lakely B. Nguyen H.M. Yang Y.C. da Costa R.M.G. Kaipainen A. Coleman R. Higano C.S. Yu E.Y. Cheng H.H. Mostaghel E.A. et al.Molecular profiling stratifies diverse phenotypes of treatment-refractory metastatic castration-resistant prostate cancer.J. Clin. Invest. 2019; 129: 4492-4505Google Scholar). About 10 to 20% of enzalutamide-resistant prostate cancers show neuroendocrine (NE) features and no effective treatments are currently available for this type of aggressive and highly invasive prostate cancer (10Davies A.H. Beltran H. Zoubeidi A. Cellular plasticity and the neuroendocrine phenotype in prostate cancer.Nat. Rev. Urol. 2018; 15: 271-286Google Scholar, 11Beltran H. Hruszkewycz A. Scher H.I. Hildesheim J. Isaacs J. Yu E.Y. Kelly K. Lin D. Dicker A. Arnold J. Hecht T. Wicha M. Sears R. Rowley D. White R. et al.The role of lineage plasticity in prostate cancer therapy resistance.Clin. Cancer Res. 2019; 25: 6916-6924Crossref Scopus (91) Google Scholar, 12Handle F. Prekovic S. Helsen C. Van den Broeck T. Smeets E. Moris L. Eerlings R. Kharraz S.E. Urbanucci A. Mills I.G. Joniau S. Attard G. Claessens F. Drivers of AR indifferent anti-androgen resistance in prostate cancer cells.Sci. Rep. 2019; 9: 13786Google Scholar). Though the continued growth and metastasis of the heavily enzalutamide-treated prostate tumors can be driven by nonandrogenic signaling, molecular underpinnings of critical targetable mechanisms in treatment-emergent NE type prostate cancer cells are yet to be fully characterized. When prostate cancer cells become resistant to strong androgen-receptor blockers, such as enzalutamide, their common characteristics change from slow-growing and noninvasive to fast-growing, highly invasive type, but the knowledge about critical signaling mechanisms driving rapid growth and resistance to enzalutamide is still limited. To better understand the mechanistic basis behind enzalutamide resistance, we developed an in vitro model by chronically treating human LNCaP, MDA-PCa-2B, and LAPC4 prostate cancer cells (AR-signaling intact) with gradually increasing doses of enzalutamide (up to 30 μM) for >12 weeks to mimic the clinical conditions in standard long-term enzalutamide therapy (13Monga J. Subramani D. Bharathan A. Ghosh J. Pharmacological and genetic targeting of 5-lipoxygenase interrupts c-Myc oncogenic signaling and kills enzalutamide-resistant prostate cancer cells via apoptosis.Sci. Rep. 2020; 10: 6649Google Scholar). The resultant cells (LNCaP-ENR, PCa-2B-ENR, and LAPC4-ENR) are completely resistant to clinically relevant doses of enzalutamide, the blood level of which goes up to ∼34 μM in average (14Gibbons J.A. Ouatas T. Krauwinkel W. Ohtsu Y. van der Walt J.S. Beddo V. de Vries M. Mordenti J. Clinical pharmacokinetic studies of enzalutamide.Clin. Pharmacokinet. 2015; 54: 1043-1055Google Scholar, 15Gibbons J.A. Vries M. Krauwinkel W. Ohtsu Y. Noukens J. van der Walt V. Mol R. Mordenti J. Ouatas T. Pharmacokinetic drug interaction studies with enzalutamide.Clin. Pharmacokinet. 2015; 54: 1057-1069Google Scholar). By gene expression array, RT-PCR, and Western blot, we found that enzalutamide-resistant prostate cancer cells overexpress Tribbles 2 (TRIB2), a member of the Tribbles pseudokinase family (TRIB1-3). We also found that overexpression of TRIB2 alone, by gene transfection, can confer resistance to physiological doses of enzalutamide. Interestingly, molecular characterization revealed that the overexpression of TRIB2 suppresses luminal characteristics and induces NE features involving the master neuronal transcription factor, BRN2 (Brain-2), and the stemness regulator, SOX2 (SRY-box 2). Moreover, inhibition of either TRIB2 or its targets (BRN2 or SOX2) resensitizes resistant cells to enzalutamide. These findings indicate that TRIB2 is a new biomarker for NE-type prostate cancer and suggest that TRIB2 may contribute to enzalutamide resistance in prostate cancer cells, at least in part, by promoting lineage plasticity and phenotype switching. A comprehensive gene expression array analysis revealed that the TRIB2 pseudokinase is grossly overexpressed in enzalutamide-resistant (EN1 and EN2) prostate cancer cells, compared to parental enzalutamide sensitive (LN1 and LN2) cells (Fig. 1, A–D). To confirm the array data, we detected TRIB2 mRNA expression by RT-PCR, and Western blot showed strong upregulation of TRIB2 protein levels in enzalutamide-resistant (LNCaP-ENR, PCa-2B-ENR, and LAPC4-ENR) prostate cancer cells (Fig. 1, E and F). Overexpression of TRIB2 is correlated with activation of the canonical Akt-signaling module showing increased phosphorylation of Akt (pSer-473) and increased protein level of Bcl-xL, which are standard markers that promote cell survival and decrease apoptosis in a variety of cells. Consistent with our results, we also found elevated mRNA levels of TRIB2 in gene expression datasets in recognized enzalutamide-resistant prostate cancer cells (Fig. S1) (16Bishop J.L. Thaper D. Vahid S. Davies A. Ketola K. Kuruma H. Jama R. Nip K.M. Angeles A. Johnson F. Wyatt A.W. Fazli L. Gleave M.E. Lin D. Rubin M.A. et al.The master neural transcription factor BRN2 is an androgen receptor-suppressed driver of neuroendocrine differentiation in prostate cancer.Cancer Discov. 2017; 7: 54-71Google Scholar). Enzalutamide-resistant cells also showed increased levels of TRIB2 protein compared to enzalutamide-sensitive cells (Fig. S2). Increased mRNA expression of TRIB2 (denoted as GS3955) was observed previously by Bisoffi et al. (17Bisoffi M. Klima I. Gresko E. Durfee P.N. Hines W.C. Griffith J.K. Studer U.E. Thalmann G.N. Expression profiles of androgen independent bone metastatic prostate cancer cells indicate up-regulation of the putative serine-threonine kinase GS3955.J. Urol. 2004; 172: 1145-1150Google Scholar), in the androgen-resistant (C4-2 and PC3) prostate cancer cells compared to androgen-sensitive (LNCaP) cells. To verify whether the in vitro observation of TRIB2 overexpression is valid in vivo, we comprehensively analyzed prostate tumors in tissue microarrays (TMAs). We found that patient-derived xenografts of prostate tumors overexpress TRIB2 when the mice were treated with enzalutamide at 30 mg/kg/day for 6 weeks (Fig. 1, G and H). Moreover, we found that a vast majority of the clinically advanced metastatic prostate tumors from enzalutamide-treated patients show a robust increase in the expression of TRIB2 proteins (Fig. 1, I and J). Altogether, these findings suggest that triggering of TRIB2 overexpression is a fundamental mechanism in prostate cancer cells both in vitro and in vivo when treated with enzalutamide, a second-generation direct inhibitor of AR activity. Note: To confirm the specificity of TRIB2 antibodies, we used a panel of TRIB2-negative and TRIB2-positive cell lines and tumor tissues and analyzed by both Western blot as well as immunohistochemistry (Fig. S3). Because overexpression of TRIB2 was observed in prostate cancer cells upon enzalutamide treatment both in vitro and in vivo, we asked the question whether TRIB2 plays any role in enzalutamide-resistant cells. By shRNA-mediated knockdown we found that downregulation of TRIB2 strongly inhibits the viability and growth of enzalutamide-resistant cells, whereas benign prostatic hyperplasia (BPH-1) cells remained unaffected (Figs. 2, A–C and S4), suggesting that TRIB2 plays a critical but selective role in enzalutamide-resistant prostate cancer cells. We also found that TRIB2 siRNA decreased LNCaP-ENR tumor growth in nude mice (Fig. 2, D and E). No specific, target-validated inhibitor of TRIB2 is commercially available. However, recently, it was demonstrated that the EGFR kinase inhibitor, Afatinib (AFA), destabilizes TRIB2 protein by covalent modification and primes TRIB2 for degradation at high micromolar doses (18Foulkes D.M. Byrne D.P. Yeung W. Shrestha S. Bailey F.P. Ferries S. Eyers C.E. Keeshan K. Wells C. Drewry D.H. Zuercher W.J. Kannan N. Eyers P.A. Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells.Sci. Signal. 2018; 11eaat7951Google Scholar). Thus, we also examined the effect of AFA and found that it strongly downregulates TRIB2 protein level and kills enzalutamide-resistant prostate cancer cells by triggering apoptosis (Fig. 2, F–H). These findings indicate that TRIB2 plays an important role in enzalutamide-resistant cells and suggest that suitable more selective TRIB2-targeting agents could be developed to kill enzalutamide-resistant prostate cancer cells. Because TRIB2 plays a critical role in the viability of enzalutamide-resistant prostate cancer cells, it has emerged as a new molecular target for therapeutic development. It also raised the question whether overexpression of TRIB2 provides any growth advantage or plays an active role in resistant prostate cancer cells for their defense against enzalutamide therapy. To address this, we transfected LNCaP and PCa-2B cells with full-length human TRIB2 gene and found that the TRIB2-overexpressing (TRIB2-OE) cells (LNCaP-TRIB2 and PCa-2B-TRIB2) show increased levels of prosurvival proteins, p-Akt and Bcl-xL, and decreased level of Forkhead Box O3, a tumor suppressor (Fig. 3A). TRIB2-OE cells also showed increased number and size of colonies (Fig. 3B) and increased capability to invade through Matrigel in Boyden chambers (Fig. 3C), suggesting that TRIB2 may play a major role as a driver to promote aggressive behavior which is frequently observed in antiandrogenic therapy-resistant prostate cancer cells. We also found that overexpression of TRIB2 alone makes prostate cancer cells resistant to therapeutic doses of enzalutamide, and the enzalutamide-resistant cells are not sensitive to the synthetic androgen, R1881 (Figs. 3D and S5). Interestingly, this resistance is abolished, and the TRIB2-OE cells become sensitive to enzalutamide again when treated with TRIB2-shRNA or AFA (Fig. 3E). Similar resensitization to enzalutamide was also observed in LNCaP-ENR and PCa-2B-ENR cells when TRIB2 was inhibited (Fig. 3, F–H). We also found that the naturally occurring castration-resistant prostate cancer cells, C4-2B, reverted to enzalutamide-sensitive state upon TRIB2 knockdown (Fig. S6). TRIB2-OE cells form larger soft-agar colonies and show no sensitivity to enzalutamide (Fig. 3I). Furthermore, we found that overexpression of TRIB2 enhances prostate tumor growth in nude mice and these tumors grow uninterrupted with enzalutamide treatment (Fig. 3, J and K). These findings suggest that not only TRIB2 enhances the aggressive characteristics, but also greatly contributes to the enzalutamide resistance mechanism in prostate cancer cells. How TRIB2 confers resistance to enzalutamide is an intriguing question. Resistance to androgen-signaling blockers may happen because of the overexpression or mutation of the AR gene, or due to development of mechanism(s) independent of androgenic signaling. To address the downstream mechanism of TRIB2 action in enzalutamide resistance, we found that TRIB2-OE enzalutamide-resistant cells (LNCaP-TRIB2 and PCa-2B-TRIB2) show a decrease in the protein level of luminal markers (cytokeratin 8 (CK8) and AR), but there is a strong increase in the expression levels of NE markers, such as Synaptophysin (SYP), Enolase-2 (NSE), and Chromogranin-A (CHGA) (Fig. 4, A and B). These findings indicated that TRIB2-induced resistance to enzalutamide does not involve reactivation of the AR function or maintenance of the luminal features, rather TRIB2 appears to be involved in transdifferentiation of luminal epithelial cells to develop NE-like characteristics (Fig. 4C). Transcriptomic analysis of enzalutamide-resistant prostate cancer cells by us and others (16Bishop J.L. Thaper D. Vahid S. Davies A. Ketola K. Kuruma H. Jama R. Nip K.M. Angeles A. Johnson F. Wyatt A.W. Fazli L. Gleave M.E. Lin D. Rubin M.A. et al.The master neural transcription factor BRN2 is an androgen receptor-suppressed driver of neuroendocrine differentiation in prostate cancer.Cancer Discov. 2017; 7: 54-71Google Scholar) also revealed a negative correlation between the expression of TRIB2 and AR signaling-associated genes (Fig. S7). Notably, we found a strong upregulation of the neuronal transcription factor, BRN2, and the stemness transcription factor, SOX2, in TRIB2-OE enzalutamide-resistant cells (Figs. 4, A and B and S8), which were characterized to promote lineage plasticity in prostate cancer cells (16Bishop J.L. Thaper D. Vahid S. Davies A. Ketola K. Kuruma H. Jama R. Nip K.M. Angeles A. Johnson F. Wyatt A.W. Fazli L. Gleave M.E. Lin D. Rubin M.A. et al.The master neural transcription factor BRN2 is an androgen receptor-suppressed driver of neuroendocrine differentiation in prostate cancer.Cancer Discov. 2017; 7: 54-71Google Scholar, 19Mu P. Zhang Z. Benelli M. Karthaus W.R. Hoover E. Chen C.C. Wongvipat J. Ku S.Y. Gao D. Cao Z. Shah N. Adams E.J. Abida W. Watson P.A. Prandi D. et al.SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer.Science. 2017; 355: 84-88Google Scholar). The regulation of NE and stemness markers by TRIB2 was confirmed by shRNA knockdown of TRIB2 in TRIB2-OE cells (Fig. 4D). We also found overexpression of NE markers and downregulation of AR in TRIB2-OE cells in tumor xenografts in mice (Fig. 4, E and F). Moreover, we found consistent strong expression of TRIB2 in multiple standard NE-type prostate tumor samples (Figs. S9 and S10). Both the Beltran and Labrecque databases show overexpression of TRIB2 in NE-type prostate cancers compared to adenocarcinoma (Fig. 4, G and H). Interestingly, inhibition of either BRN2 or SOX2 resensitizes TRIB2-OE cells to enzalutamide treatment, indicating that the molecular mechanism of TRIB2 may involve upregulation of BRN2 and SOX2, presumably to increase cellular plasticity (Fig. S11). Altogether, our findings suggest that TRIB2 helps prostate cancer cells to evade enzalutamide therapy, apparently by switching their identity from luminal to assume NE characteristics. Thus, it appears that a strong positive correlation exists between TRIB2 overexpression and the development of NE features in prostate cancer cells. Targeting of androgenic signaling has been improved significantly due to the introduction of strong AR antagonists, though the benefit is temporary, and resistance invariably develops which mostly occurs within 5 years since therapy begins (5Blatt E.B. Raj G.V. Molecular mechanisms of enzalutamide resistance in prostate cancer.Cancer Drug Resist. 2019; 2: 189-197Google Scholar, 6Chen W.S. Aggarwal R. Zhang L. Zhao S.G. Thomas G.V. Beer T.M. Quigley D.A. Foye A. Playdle D. Huang J. Lloyd P. Lu E. Sun D. Guan X. Rettig M. et al.Genomic drivers of poor prognosis and enzalutamide resistance in metastatic castration-resistant prostate cancer.Eur. Urol. 2019; 76: 562-571Google Scholar, 7Akamatsu S. Inoue T. Ogawa O. Gleave M.E. Clinical and molecular features of treatment-related neuroendocrine prostate cancer.Int. J. Urol. 2018; 25: 345-351Google Scholar). It has been realized that enzalutamide-resistant prostate cancer frequently assumes deadly phenotype if AR-independent mechanisms develop, though the spectrum of which is yet to be fully characterized. We observed that overexpression of TRIB2 occurs upon treatment with enzalutamide, both in vitro and in vivo (Fig. 1). Our findings of the gross overexpression of TRIB2 by enzalutamide treatment and the aggressive growth characteristics of TRIB2-OE cells signify a negative impact of AR blockade therapy for prostate cancer. Though upregulation of TRIB2 in prostate cancer cells upon enzalutamide treatment is a remarkable finding, why and how TRIB2 is overexpressed in these cells is not known at this time. Of the multitude of factors playing roles in enzalutamide resistance, derepression of AR-controlled suppression of gene expression seems to be one. We found that PC3 and DU145 prostate cancer cells that are naturally deficient in AR activity express high levels of TRIB2 protein. Interestingly, forced overexpression and activation of full-length AR downregulates TRIB2 protein level, suggesting a negative regulation of TRIB2 by AR (Fig. S12). Negative regulation of the neuronal transcription factor (BRN2) and SPINK by AR has been found recently (16Bishop J.L. Thaper D. Vahid S. Davies A. Ketola K. Kuruma H. Jama R. Nip K.M. Angeles A. Johnson F. Wyatt A.W. Fazli L. Gleave M.E. Lin D. Rubin M.A. et al.The master neural transcription factor BRN2 is an androgen receptor-suppressed driver of neuroendocrine differentiation in prostate cancer.Cancer Discov. 2017; 7: 54-71Google Scholar, 20Tiwari R. Manzar N. Bhatia V. Yadav A. Nengroo M.A. Datta D. Carskadon S. Gupta N. Sigouros M. Khani F. Poutanen M. Zoubeidi A. Beltran H. Palanisamy N. Ateeq B. Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer.Nat. Commun. 2020; 11: 1-19Google Scholar). The aggressive nature of prostate cancer cells post-enzalutamide therapy, correlates well with aggressive melanoma and lung cancer cells which overexpress TRIB2. This could be, at least in part, because of the absence of strong androgenic signaling in skin and lung cells, the presence of which may inhibit the expression of TRIB2 in prostate cells. However, further work is needed to substantiate this notion. Curiosity lingered around the consequence of TRIB2 overexpression in prostate cancer cells upon enzalutamide treatment. TRIB2 was originally discovered in Drosophila as a regulator of wing pattern (21Mata J. Curado S. Ephrussi A. Rorth P. Tribbles coordinates mitosis and morphogenesis in Drosophila by regulating string/CDC25 proteolysis.Cell. 2000; 101: 511-522Google Scholar). Later, its expression in human and other organisms as well as a role in cancer cells were observed (22Hill R. Madureira P.A. Ferreira B. Baptista I. Machado S. Colaco L. Dos Santos M. Liu N. Dopazo A. Ugurel S. Adrienn A. Kiss-Toth E. Isbilen M. Gure A.O. Link W. TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT.Nat. Commun. 2017; 8: 14687Google Scholar, 23Eyers P.A. Keeshan K. Kannan N. Tribbles in the 21st century: The evolving roles of tribbles pseudokinases in biology and disease.Trends Cell Biol. 2017; 27: 284-298Google Scholar). We wanted to address whether TRIB2 plays any role in enzalutamide-resistant prostate cancer cells. Interestingly, we observed that treatment with TRIB2-shRNA decreased TRIB2 protein level and proportionately reduced the viability of enzalutamide-resistant cells, suggesting that TRIB2 plays a critical role in the enzalutamide resistance mechanism in prostate cancer cells (Fig. 2). That overexpression of TRIB2 alone can confer complete resistance to physiological doses of enzalutamide is particularly interesting (Fig. 3). However, a more remarkable feature recently revealed from our work, is the resistance of TRIB2-OE prostate cancer cells also to other second-generation anti-androgens that are frequently used in the clinic in addition to enzalutamide, such as apalutamide, darolutamide, and abiraterone (not shown). Intriguingly, enzalutamide-resistant TRIB2-OE cells show decreased protein level of luminal markers (CK8 and AR) and increased the level of NE markers (BRN2, SYP, NSE, and CHGA), and these characteristics are reversed when TRIB2 is downregulated by shRNA (Fig. 4, A and D). These findings suggest that TRIB2 is a driver for transdifferentiation of prostate cancer cells from luminal to NE type. An AR activity low stemness program as well as NE differentiation were observed in enzalutamide-resistant prostate cancer cells and tumors (8Beltran H. Prandi D. Mosquera J.M. Benelli M. Puca L. Cyrta J. Marotz C. Giannopoulou E. Chakravarthi B.V. Varambally S. Tomlins S.A. Nanus D.M. Tagawa S.T. Van Allen E.M. Elemento O. et al.Divergent clonal evolution of castration-resistant neuroendocrine prostate cancer.Nat. Med. 2016; 22: 298-305Google Scholar, 9Labrecque M.P. Coleman I.M. Brown L.G. True L.D. Kollath L. Lakely B. Nguyen H.M. Yang Y.C. da Costa R.M.G. Kaipainen A. Coleman R. Higano C.S. Yu E.Y. Cheng H.H. Mostaghel E.A. et al.Molecular profiling stratifies diverse phenotypes of treatment-refractory metastatic castration-resistant prostate cancer.J. Clin. Invest. 2019; 129: 4492-4505Google Scholar, 10Davies A.H. Beltran H. Zoubeidi A. Cellular plasticity and the neuroendocrine phenotype in prostate cancer.Nat. Rev. Urol. 2018; 15: 271-286Google Scholar, 11Beltran H. Hruszkewycz A. Scher H.I. Hildesheim J. Isaacs J. Yu E.Y. Kelly K. Lin D. Dicker A. Arnold J. Hecht T. Wicha M. Sears R. Rowley D. White R. et al.The role of lineage plasticity in prostate cancer therapy resistance.Clin. Cancer Res. 2019; 25: 6916-6924Crossref Scopus (91) Google Scholar, 12Handle F. Prekovic S. Helsen C. Van den Broeck T. Smeets E. Moris L. Eerlings R. Kharraz S.E. Urbanucci A. Mills I.G. Joniau S. Attard G. Claessens F. Drivers of AR indifferent anti-androgen resistance in prostate cancer cells.Sci. Rep. 2019; 9: 13786Google Scholar, 24Alumkal J.J. Suna D. Lua E. Beer T.M. Thomas G.V. Latour E. Aggarwal R. Cetnar J. Ryan C.J. Tabatabaei S. Bailey S. Turina C.B. Quigley D.A. Guan X. Foye A. et al.Transcriptional profiling identifies an androgen receptor activity-low, stemness program associated with enzalutamide resistance.Proc. Natl. Acad. Sci.U. S. A. 2020; 117: 12315-12323Google Scholar, 25Aggarwal R. Huang J. Alumkal J.J. Zhang L. Feng F.Y. Thomas G.V. Weinstein A.S. Friedl V. Zhang C. Witte O.N. Lloyd P. Gleave M. Evans C.P. Youngren J. Beer T.M. et al.Small, clinical and genomic characterization of treatment-emergent small-cell neuroendocrine prostate cancer: A multi-institutional prospective study.J. Clin. Oncol. 2018; 36: 2492-2503Google Scholar). Recently, the SOX2-mediated lineage plasticity and development of NE phenotype have been demonstrated in PTEN (−/−): Rb1 (−/−) double knockout mice (19Mu P. Zhang Z. Benelli M. Karthaus W.R. Hoover E. Chen C.C. Wongvipat J. Ku S.Y. Gao D. Cao Z. Shah N. Adams E.J. Abida W. Watson P.A. Prandi D. et al.SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer.Science. 2017; 355: 84-88Google Scholar, 26Ku S.Y. Rosario S. Wang Y. Mu P. Seshadri M. Goodrich Z.W. Goodrich M.M. Labbé D.P. Gomez E.C. Wang J. Long H.W. Xu B. Brown M. Loda M. Sawyers C.L.