Prostate Tumorigenesis Research Articles

Personalized Medicine for Prostate Cancer: Is Targeting Metabolism a Reality?

Prostate cancer invokes major shifts in gene transcription and metabolic signaling to mediate alterations in nutrient acquisition and metabolic substrate selection when compared to normal tissues. Exploiting such metabolic reprogramming is proposed to enable the development of targeted therapies for prostate cancer, yet there are several challenges to overcome before this becomes a reality. Herein, we outline the role of several nutrients known to contribute to prostate tumorigenesis, including fatty acids, glucose, lactate and glutamine, and discuss the major factors contributing to variability in prostate cancer metabolism, including cellular heterogeneity, genetic drivers and mutations, as well as complexity in the tumor microenvironment. The review draws from original studies employing immortalized prostate cancer cells, as well as more complex experimental models, including animals and humans, that more accurately reflect the complexity of the in vivo tumor microenvironment. In synthesizing this information, we consider the feasibility and potential limitations of implementing metabolic therapies for prostate cancer management.

Stage-specific differential expression of zinc transporter SLC30A and SLC39A family proteins during prostate tumorigenesis.

Prostate cancer (PCa) initiation and progression uniquely modify the prostate milieu to aid unrestrained cell proliferation. One salient modification is the loss of the ability of prostate epithelial cells to accumulate high concentrations of zinc; however, molecular alterations associated with loss of zinc accumulating capability in malignant prostate cells remain poorly understood. Herein, we assessed the stage-specific expression of zinc transporters (ZNTs) belonging to the ZNT(SLC30A) and Zrt- and Irt-like protein (ZIP) (SLC39A) solute-carrier family in the prostate tissues of different genetically engineered mouse models (GEMM) of PCa (TMPRSS2-ERG.Ptenflox/flox , Hi-Myc+/- , and transgenic adenocarcinoma ofmouseprostate), their age-matched wild-type controls, and 104 prostate core biopsies from human patients with different pathological lesions. Employing immunohistochemistry, differences in the levels of protein expression and spatial distribution of ZNT were evaluated as a function of the tumor stage. Results indicated that the expression of zinc importers (ZIP1, ZIP2, and ZIP3), which function to sequester zinc from circulation and prostatic fluid, was low to negligible in the membranes of the malignant prostate cells in both GEMM and human prostate tissues. Regarding zinc exporters (ZNT1, ZNT2, ZNT9, and ZNT10) that export excess zinc into the extracellular spaces or intracellular organelles, their expression was low in normal prostate glands of mice and humans; however, it was significantly upregulated in prostate adenocarcinoma lesions in GEMM and PCa patients. Together, our findings provide new insights into altered expression of ZNTs during the progression of PCa and indicate that changes in zinc homeostasis could possibly be an early-initiation event during prostate tumorigenesis and a likely prevention/intervention target.

Abstract PO-134: Differential effects of CXCR1 and CXCR2 receptors on prostate tumorigenesis

Abstract Chemokines and their receptors are important proteins that promote growth of various malignant tumors. In prostate cancer, some members of the CXC chemokine receptor have been shown to enhance angiogenesis, proliferation and metastasis. In this study we assess the roles of the interleukin-8 (IL-8/CXCL8) receptors CXCR1 and CXCR2 in prostate cancer development, using the African American MDA PCa 2b cell line. Our results show that overexpression of CXCR2 enhanced in-vitro cell proliferation, soft agar growth and in-vivo tumor xenograft in nude mice, whereas overexpression of CXCR1 inhibited cell proliferation and tumor growth, relative to control cells. Interestingly, cells overexpressing CXCR1 exhibited a more mesenchymal phenotype, characterized by reduced E-Cadherin but increased N-Cadherin and vimentin protein expression. CXCR1 overexpression also blocked AKT activation and signal transduction. CXCR2 overexpression, however, resulted in a neuroendocrine phenotype, characterized by reduced androgen (AR) receptor expression and altered chromogranin A expression. In conclusion, these results indicate that CXCR1 and CXCR2 overexpression may play alternate roles on prostate tumorigenesis. Citation Format: Timothy O. Adekoya, Nikia Smith, Parag Kothari, Ricardo M. Richardson. Differential effects of CXCR1 and CXCR2 receptors on prostate tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-134.

Fibroblast heterogeneity in prostate carcinogenesis.

Our understanding of stromal components, specifically cancer-associated fibroblasts (CAF), in prostate cancer (PCa), has evolved from considering these cells as inert bystanders to acknowledging their significance as players in prostate tumorigenesis. CAF are multifaceted-they promote cancer cell growth, migration and remodel the tumor microenvironment. Although targeting CAF could be a promising strategy for PCa treatment, they incorporate a high but undefined degree of intrinsic cellular heterogeneity. The interaction between CAF subpopulations, with the normal and tumor epithelium and with other cell types is not yet characterized. Defining these interactions and the critical signaling nodes that support tumorigenesis will enable the development of novel strategies to control prostate cancer progression. Here we will discuss the origins, molecular and functional heterogeneity of CAF in PCa. We highlight the challenges associated with delineating CAF heterogeneity and discuss potential areas of research that would assist in expanding our knowledge of CAF and their role in PCa tumorigenesis.

Novel Therapeutic Targets and Biomarkers Associated with Prostate Cancer-Associated Fibroblasts (CAFs).

Despite advances in treatment, prostate cancer remains a significant cause of morbidity and mortality worldwide. While the vast majority of prostate cancer research has centered on malignant epithelial cells, the tumor mi croenvironment (TME) has recently become increasingly recognized as an important regulator of tumor progression and response to treatment. Among the diverse cell types within the TME, stromal fibroblasts, in particular cancer-associated fibroblasts (CAFs), play an important role in prostate cancer progression. This is highlighted by the prognostic value of CAF markers in prostate cancer, which can predict disease recurrence, metastasis, and patient survival. There are also an increasing number of studies that demonstrate the critical role of CAFs in mediating response to specific therapies and CAF signaling pathways as potential therapeutic targets. However, further investigation into the mechanisms that underpin the interactions between cancer cells and CAFs are required to develop novel therapeutic approaches and identify predictive and prognostic biomarkers in CAFs. In this review, we discuss the current knowledge of CAF-dependent regulatory pathways in prostate tumorigenesis and their prognostic and therapeutic potential. Furthermore, we explore the emerging models and technologies that are likely to progress this field of research in terms of discovery and translation to the clinic.

The Effect of Oncogene Proteins of Human Papillomaviruses on Apoptosis Pathways in Prostate Cancer

The ability of host cells to activate apoptosis is perhaps the most potent weapon for helping cells eliminate viruses. Human papillomaviruses (HPV) activate several pathways, enabling the infected cells to avoid extrinsic and intrinsic apoptosis pathways. The incapacity of prostatic epithelial cells to induce apoptosis leads to the invasive development of prostate cancer. For the pathogenesis of prostate cancer, several risk factors have been reported; for example, some viruses and infectious diseases have been proposed as causative agents for their relation to prostate diseases. According to several studies, high-risk human papillomaviruses cause malignancy by interfering with the apoptotic and inflammatory pathways; these viruses, such as HPV16 and HPV18, block apoptotic pathways and result in prostate cancer. This review is dedicated to presenting a summary of oncogenes (E5, E6, and E7) HR-HPVs’ functions on signaling pathways, inflammation in prostate tumorigenesis, and emphasizing the link between these oncogenes with apoptosis and prostate cancer.

E2F transcription factor 2-activated DLEU2 contributes to prostate tumorigenesis by upregulating serum and glucocorticoid-induced protein kinase 1

Long noncoding RNAs (lncRNAs) participate in biological processes in multiple types of tumors. However, the regulatory patterns of lncRNAs in prostate cancer remain largely unclear. Here, we evaluated the expression and roles of the lncRNA DLEU2 in prostate cancer. Our results showed that DLEU2 was upregulated in advanced prostate cancer tissues. Patients with prostate cancer displaying high expression of DLEU2 had a poor prognosis. Moreover, we demonstrated that overexpression of DLEU2 facilitated the proliferation, migration, and invasion of prostate cancer in vitro. Mechanistically, DLEU2 promoted serum and glucocorticoid-induced protein kinase 1 (SGK1) expression by acting as an miR-582-5p sponge, and the transcription of DLEU2 was activated by the dysregulation of E2F transcription factor 2 (E2F2) expression in prostate cancer. Furthermore, knockdown of DLEU2 attenuated prostate cancer tumorigenesis in vivo. Notably, these findings suggested that E2F2-activated DLEU2 may function as a competing endogenous RNA to facilitate prostate cancer progression by targeting the miR-582-5p/SGK1 axis.

MicroRNA-21 deficiency suppresses prostate cancer progression through downregulation of the IRS1-SREBP-1 signaling pathway.

Sterol regulatory element-binding protein 1 (SREBP-1), a master transcription factor in lipogenesis and lipid metabolism, is critical for disease progression and associated with poor outcomes in prostate cancer (PCa) patients. However, the mechanism of SREBP-1 regulation in PCa remains elusive. Here, we report that SREBP-1 is transcriptionally regulated by microRNA-21 (miR-21) in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of SREBP-1, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) in Pten/Trp53 double-null mouse embryonic fibroblasts (MEFs) and Pten/Trp53 double-null mutant mice. Strikingly, miR-21 loss significantly reduced cell proliferation and suppressed the prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, miR-21 inactivation decreased the levels of SREBP-1, FASN, and ACC in human PCa cells through downregulation of insulin receptor substrate 1 (IRS1)-mediated transcription and induction of cellular senescence. Conversely, miR-21 overexpression increased cell proliferation and migration; as well as the levels of IRS1, SREBP-1, FASN, and ACC in human PCa cells. Our findings reveal that miR-21 promotes PCa progression by activating the IRS1/SREBP-1 axis, and targeting miR-21/SREBP-1 signaling pathway can be a novel strategy for controlling PCa malignancy.

Transcriptional regulation and ubiquitination-dependent regulation of HnRNPK oncogenic function in prostate tumorigenesis

BackgroundHeterogeneous nuclear ribonucleoprotein K (HnRNPK) is a nucleic acid-binding protein that regulates diverse biological events. Pathologically, HnRNPK proteins are frequently overexpressed and clinically correlated with poor prognosis in various types of human cancers and are therefore pursued as attractive therapeutic targets for select patients. However, both the transcriptional regulation and degradation of HnRNPK in prostate cancer remain poorly understood.MethodsqRT-PCR was used to detect the expression of HnRNPK mRNA and miRNA; Immunoblots and immunohistochemical assays were used to determine the levels of HnRNPK and other proteins. Flow cytometry was used to investigate cell cycle stage. MTS and clonogenic assays were used to investigate cell proliferation. Immunoprecipitation was used to analyse the interaction between SPOP and HnRNPK. A prostate carcinoma xenograft mouse model was used to detect the in vivo effects of HnRNPK and miRNA.ResultsIn the present study, we noted that HnRNPK emerged as an important player in the carcinogenesis process of prostate cancer. miR-206 and miR-613 suppressed HnRNPK expression by targeting its 3’-UTR in PrCa cell lines in which HnRNPK is overexpressed. To explore the potential biological function, proliferation and colony formation of PrCa cells in vitro and tumor growth in vivo were also dramatically suppressed upon reintroduction of miR-206/miR-613. We have further provided evidence that Cullin 3 SPOP is a novel upstream E3 ubiquitin ligase complex that governs HnRNPK protein stability and oncogenic functions by promoting the degradation of HnRNPK in polyubiquitination-dependent proteolysis in the prostate cancer setting. Moreover, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of HnRNPK proteins.ConclusionOur findings reveal new posttranscriptional and posttranslational modification mechanisms of HnRNPK regulation via miR-206/miR-613 and SPOP, respectively. More importantly, given the critical oncogenic role of HnRNPK and the high frequency of SPOP mutations in prostate cancer, our results provide a molecular rationale for the clinical investigation of novel strategies to combat prostate cancer based on SPOP genetic status.

Loss of FBXO31-mediated degradation of DUSP6 dysregulates ERK and PI3K-AKT signaling and promotes prostate tumorigenesis.

SUMMARYFBXO31 is the substrate receptor of one of many CUL1-RING ubiquitin ligase (CRL1) complexes. Here, we show that low FBXO31 mRNA levels are associated with high pre-operative prostate-specific antigen (PSA) levels and Gleason grade in human prostate cancer. Mechanistically, the ubiquitin ligase CRL1FBXO31 promotes the ubiquitylation-mediated degradation of DUSP6, a dual specificity phosphatase that dephosphorylates and inactivates the extracellular-signal-regulated kinase-1 and −2 (ERK1/2). Depletion of FBXO31 stabilizes DUSP6, suppresses ERK signaling, and activates the PI3K-AKT signaling cascade. Moreover, deletion of FBXO31 promotes tumor development in a mouse orthotopic model of prostate cancer. Treatment with BCI, a small molecule inhibitor of DUSP6, suppresses AKT activation and prevents tumor formation, suggesting that the FBXO31 tumor suppressor activity is dependent on DUSP6. Taken together, our studies highlight the relevance of the FBXO31-DUSP6 axis in the regulation of ERK- and PI3K-AKT-mediated signaling pathways, as well as its therapeutic potential in prostate cancer.

SPOP promotes CDCA5 degradation to regulate prostate cancer progression via the AKT pathway.

The E3 ubiquitin ligase adaptor Speckle-type POZ protein (SPOP) plays an important tumour suppressor role in prostate cancers (PCa), with mutation rate up to 15%. However, how SPOP mutations regulate prostate tumorigenesis remains elusive. Here, we report the identification of cell division cycle associated 5 (CDCA5) as a SPOP substrate. We found that SPOP interacts with CDCA5 and promotes its polyubiquitin degradation in a degron-dependent manner. This effect was greatly impaired by introducing PCa associated SPOP mutations. Importantly, we found that CDCA5 was essential for PCa cells to survive and proliferate. CDCA5 depletion in PCa cells led to cessation of proliferation, G2M arrest, severe sister chromatid aggregation disturbance, and apoptosis. we also found that CDCA5 knockdown decreased the protein expression of p-GSK3β, increased the activity of caspase-3, caspase-9, and the Bax/Bcl-2 ratio. Besides, we confirmed that CDCA5 interrupted cancer cell behavior via the AKT pathway. In contrast, silencing SPOP or overexpressing CDCA5 increased cell proliferation. Consistently, depleting SPOP along with CDCA5, or overexpressing CDCA5 along with SPOP also caused the growth of cells repressed. Consistent with the functional role of CDCA5, the mRNA and protein levels of CDCA5 were significantly increased in PCa, compared to normal tissues, and its high expression was associated with more severe lymph node metastasis, higher Gleason score, and poorer prognosis. Together, our data showed that SPOP plays a crucial role in inhibiting tumorigenesis and partly achieved this by promoting the degradation of oncoprotein CDCA5.

CdGAP promotes prostate cancer metastasis by regulating epithelial-to-mesenchymal transition, cell cycle progression, and apoptosis

High mortality of prostate cancer patients is primarily due to metastasis. Understanding the mechanisms controlling metastatic processes remains essential to develop novel therapies designed to prevent the progression from localized disease to metastasis. CdGAP plays important roles in the control of cell adhesion, migration, and proliferation, which are central to cancer progression. Here we show that elevated CdGAP expression is associated with early biochemical recurrence and bone metastasis in prostate cancer patients. Knockdown of CdGAP in metastatic castration-resistant prostate cancer (CRPC) PC-3 and 22Rv1 cells reduces cell motility, invasion, and proliferation while inducing apoptosis in CdGAP-depleted PC-3 cells. Conversely, overexpression of CdGAP in DU-145, 22Rv1, and LNCaP cells increases cell migration and invasion. Using global gene expression approaches, we found that CdGAP regulates the expression of genes involved in epithelial-to-mesenchymal transition, apoptosis and cell cycle progression. Subcutaneous injection of CdGAP-depleted PC-3 cells into mice shows a delayed tumor initiation and attenuated tumor growth. Orthotopic injection of CdGAP-depleted PC-3 cells reduces distant metastasic burden. Collectively, these findings support a pro-oncogenic role of CdGAP in prostate tumorigenesis and unveil CdGAP as a potential biomarker and target for prostate cancer treatments.

Suppression of ANT2 by miR-137 Inhibits Prostate Tumorigenesis.

Prostate cancer (PCa) is a serious disease that affects men’s health. To date, no effective and long-lasting treatment option for this condition is available in clinical practice. ANT2 is highly expressed in a variety of hormone-related cancers, but its relationship and regulatory mechanism with PCa are unclear. In this study, we found that ANT2 expression was significantly upregulated in PCa tissues relative to control samples. Genetic knockdown of ANT2 effectively inhibited, while overexpression promoted, proliferation, migration, and invasion of PCa cells. In addition, miR-137 expression was reduced in prostate cancer tissues relative to control tissues. We identified a regulatory site for miR-137 in the 3′-UTR of ANT2 mRNA; luciferase reporter assays indicated that ANT2 is a direct target gene for miR-137. Transfecting cells with miR-137 mimics and/or an ANT2-encoding plasmid revealed that ANT2 promotes proliferation, migration, and invasion of PCa, whereas co-expression of miR-137 mimics inhibited these behaviors. These observations suggest that miR-137 mimics inhibit development of PCa by antagonizing expression of ANT2. Furthermore, tumorigenic assays in nude mice showed that miR-137 inhibitors abolished the inhibitory effect of ANT2 knockdown on PCa tumor growth. Collectively, our findings suggest that ANT2, a target gene of miR-137, is intimately involved in development of PCa, providing new evidence for the mechanism underlying pathogenesis of PCa as well as new options for targeted therapy.

Reshaping of the androgen-driven chromatin landscape in normal prostate cells by early cancer drivers and effect on therapeutic sensitivity.

SUMMARYThe normal androgen receptor (AR) cistrome and transcriptional program are fundamentally altered in prostate cancer (PCa). Here, we profile the chromatin landscape and AR-directed transcriptional program in normal prostate cells and show the impact of SPOP mutations, an early event in prostate tumorigenesis. In genetically normal mouse prostate organoids, SPOP mutation results in accessibility and AR binding patterns similar to that of human PCa. Consistent with dependence on AR signaling, castration of SPOP mutant mouse models results in the loss of neoplastic phenotypes, and human SPOP mutant PCa shows a favorable response to AR-targeted therapies. Together, these data validate mouse prostate organoids as a robust model for studying epigenomic and transcriptional alterations in normal prostate, provide valuable datasets for further studies, and show that a single genomic alteration may be sufficient to reprogram the chromatin of normal prostate cells toward oncogenic phenotypes, with potential therapeutic implications for AR-targeting therapies.

Abstract 2393: The systematic evaluation of the oncogenic lncRNA LINC00963 using a CRISPRScan technique

Abstract Long non-coding RNAs (LncRNAs) have been found to play important roles in maintaining and promoting cellular proliferation in cancerous cells. One such lncRNA, LINC00963 has been found to promote cellular proliferation, migration and tumorigenesis in prostate, ovarian, breast and non-small cell lung (NSCLC) cancer. In this study, we aim to identify important functional regions of this lncRNA with a view to identify unique regions for targeted drug therapy. In order to achieve this, we sought to investigate this lncRNA in the A549 NSCLC cell line. We found that LINC00963 was highly expressed and when targeted with either siRNAs or antisense oligonucleotides, cells rapidly undergo apoptosis. Using cell death as a proxy for loss-of-function, we characterized LINC00963 at the genomic level using a CRIPSRScan approach. CRISPRScan is a novel way of determining lncRNA functional domains through the introduction of local mutations. These mutations could potentially disrupt lncRNA function, which for LINC00963 would lead to apoptosis of the cell. We created a unique lentivirus library comprised of 1784 single guide RNAs spanning the length of LINC00963 in order to precisely identify regions of functional importance. Upon transduction in a stably expressing Cas9-A549 cell line, we collected cellular fractions over time, and using high-throughput sequencing (NextSeq, Illumina), evaluated the depletion of these sgRNAs as a proxy for their importance in LINC00963 function. The identification of these key genomic regions that may render LINC00963 non-functional may offer new lncRNA target sites for anti-cancer therapies in the future. Additionally, the utilization of an in-depth CRISPR-Cas9 approach to determine important lncRNA domains provides a novel and precise way in which to evaluate lncRNA function. Citation Format: Roslyn M. Ray, Lu Lang, Sheela P. Pokharel, Denis O'Meally, Tristan A. Scott, Chun-Wei Chen, Kevin V. Morris. The systematic evaluation of the oncogenic lncRNA LINC00963 using a CRISPRScan technique [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2393.

Abstract 81: Trop2 regulates prostate cancer growth and metastasis through distinct molecular mechanisms

Abstract Background: The first line of treatment for men with advanced prostate cancer is androgen deprivation therapy. Although initial responses are observed, prostate cancer commonly relapses in its lethal metastatic form referred to as castration resistant prostate cancer (CRPC) with 1-2 years mean survival time. Neuroendocrine prostate cancer (NEPC) is highly aggressive, AR independent subtype and usually emerges post castration resistance. We recently identified that the cell surface receptor, Trop2, is a new driver of NEPC. Moreover, we demonstrated that Trop2 regulates prostate cancer growth and metastasis. In this study, we set out to delineate the molecular mechanisms through which Trop2 regulates prostate cancer growth and metastasis. Methods: Proximity-dependent Biotin Identification (BioID) followed by mass spectrometry was performed to identify Trop2 interactome. Lentiviral infection was used to generate prostate cancer cell lines with over-expression of Trop2 and knock-down of NOTCH1, SLC4A7, PLEC, and OCLN shRNA to modulate gene expression levels. In vitro functional assays were performed including colony formation, and Matrigel drop 3D cell invasion assays. Results: The Trop2 membrane interactome was identified utilizing Proximity-dependent Biotin Identification (BioID) in living cells and uncovered that Trop2-mediated prostate cancer growth and metastasis are orchestrated by distinct downstream pathways including Notch signaling (NOTCH1), control of intracellular pH (SLC4A7), exosome secretion (PLEC), and tight junctions (OCLN). Interaction of Trop2 binding partners including NOTCH1, SLC4A7, PLEC and OCLN with Trop2 were further validated by fluorescence resonance energy transfer (FRET) using confocal microscopy. Moreover, knocking down the Trop2 interacting partners in Trop2 over-expressing prostate cancer cells suppressed Trop2-driven growth and invasion ability. Conclusions: In a previous study, we identified cell surface receptor, Trop2, as a novel driver of metastatic NEPC. Herein, our new findings reveal that Trop2 interacts with NOTCH1, SLC4A7, PLEC, and OCLN, which may highlight novel biological functions of Trop2 in prostate tumorigenesis and provide new understanding of the potential mechanism of neuroendocrine differentiation and metastasis to provide new therapeutic strategy for metastatic CRPC with neuroendocrine features. Citation Format: Tanya Ivanova Stoyanova, En-Chi Hsu, Shiqin Liu, Fernando Jose Garcia Marques, Abel Bermudez, Merve Aslan, Michelle Shen, Sharon Pitteri, James D. Brooks. Trop2 regulates prostate cancer growth and metastasis through distinct molecular mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 81.

Abstract 2928: Evaluating Cks1-Skp2 interface as an effective therapeutic target for RB1-deficient metastatic castration-resistant prostate cancer (mCRPC)

Abstract Most prostate cancers are locally confined and can be cured by surgery, radiation therapy, and androgen deprivation therapy (ADT). Many prostate cancers initially respond to ADT but become resistant over time and ultimately relapse with a more aggressive, chemotherapy-resistant, and often metastatic disease, termed metastatic castration-resistant prostate cancer (mCRPC), with five-year survival rate <30%. Compared with regional and castration-sensitive prostate cancer, mCRPC harbor higher frequency of inactivating RB1 mutations. Unfortunately, no targeted therapy is available for RB1-deficient tumors, as it is not possible to restore this tumor suppressor in tumor cells. Therefore, we aim to identify downstream signaling targets of pRb which could serve as drug targets and thereby restore the critical functions of pRb. Our group previously identified the E3 ubiquitin ligase SCF-Skp2 as a direct repression target of pRb. Skp2 knockout completely blocks prostate tumorigenesis and cancer-related deaths in an Rb1 and Trp53 double deficient mouse model. As a substrate of Skp2, the CDK inhibitor p27 is upregulated upon Skp2 deletion, which slows cell cycle progression and thus cell proliferation. Our research findings indicate that Skp2 is a promising target for RB1 deficient mCRPC treatment. Skp2 post-translationally regulates a large number of proteins, while Cks1 serves as an essential adaptor protein only for Skp2-mediated p21/p27/p57 ubiquitination, and its Asn45 plays a critical role in binding with Skp2. A Cks1 N45R mutant has been reported to abolish the binding of Cks1 with Skp2. In order to specifically and effectively target Skp2's function in p27 degradation, we generated Cks1 N45R knockin mice and evaluated its anti-tumorigenesis effect. Here we show Cks1 N45R knockin completely inhibits tumorigenesis in the Rb1 and Trp53 double deficient prostate cancer mouse model, with p27 being elevated in the prostate to the comparable amount of that in Skp2 knockout. Surprisingly, Cks1 N45R leads to reduced Skp2 protein stability and consequently the total protein level of Skp2 decreases by more than 50%. We found that the CDK1/2 inhibitor Dinaciclib downregulates Skp2 protein levels by promoting Skp2's interaction and ubiquitination with the E3 ligase Cdh1. Our preliminary data suggests that increased p27 facilitates Cdh1-mediated Skp2 degradation by suppressing Cdk2/CyclinA-mediated Skp2 and Cdh1 phosphorylation. This study highlights the potential of developing small molecule inhibitors targeting the Cks1-Skp2 interface for RB1-mutant mCPRC, it also shows the regulation of Skp2 protein levels may be a viable strategy to phenocopy the antitumor effect of Skp2 knockout. Furthermore, we expect that the combination of Cdk1/2 inhibitors and Cks1-Skp2 interface inhibitors would increase the efficacy and selectivity of targeted therapy in mCRPC. Citation Format: Yingjiao Xue, Liang Zhu, Hongling Zhao, Edward L. Schwartz. Evaluating Cks1-Skp2 interface as an effective therapeutic target for RB1-deficient metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2928.

HGK promotes metastatic dissemination in prostate cancer

Metastasis is the process of cancer cell dissemination from primary tumors to different organs being the bone the preferred site for metastatic homing of prostate cancer (PCa) cells. Prostate tumorigenesis is a multi-stage process that ultimately tends to advance to become metastatic PCa. Once PCa patients develop skeletal metastases, they eventually succumb to the disease. Therefore, it is imperative to identify essential molecular drivers of this process to develop new therapeutic alternatives for the treatment of this devastating disease. Here, we have identified MAP4K4 as a relevant gene for metastasis in PCa. Our work shows that genetic deletion of MAP4K4 or pharmacological inhibition of its encoded kinase, HGK, inhibits metastatic PCa cells migration and clonogenic properties. Hence, MAP4K4 might promote metastasis and tumor growth. Mechanistically, our results indicate that HGK depleted cells exhibit profound differences in F-actin organization, increasing cell spreading and focal adhesion stability. Additionally, HGK depleted cells fails to respond to TNF-α stimulation and chemoattractant action. Moreover, here we show that HGK upregulation in PCa samples from TCGA and other databases correlates with a poor prognosis of the disease. Hence, we suggest that it could be used as prognostic biomarker to predict the appearance of an aggressive phenotype of PCa tumors and ultimately, the appearance of metastasis. In summary, our results highlight an essential role for HGK in the dissemination of PCa cells and its potential use as prognostic biomarker.

Inflammatory bowel disease induces inflammatory and pre-neoplastic changes in the prostate.

Background:Inflammatory bowel disease (IBD) has been implicated as a risk factor for prostate cancer, however, the mechanism of how IBD leads to prostate tumorigenesis is not known. Here, we investigated whether chronic intestinal inflammation leads to pro-inflammatory changes associated with tumorigenesis in the prostate.Methods:Using clinical samples of men with IBD who underwent prostatectomy, we analyzed whether prostate tumors had differences in lymphocyte infiltrate compared to non-IBD controls. In a mouse model of chemically-induced intestinal inflammation, we investigated whether chronic intestinal inflammation could be transferred to the wild-type mouse prostate. In addition, mouse prostates were evaluated for activation of pro-oncogenic signaling and genomic instability.Results:A higher proportion of men with IBD had T and B lymphocyte infiltration within prostate tumors. Mice with chronic colitis showed significant increases in prostatic CD45+ leukocyte infiltration and elevation of three pro-inflammatory cytokines—TIMP-1, CCL5, and CXCL1 and activation of AKT and NF-kB signaling pathways. Lastly, mice with chronic colitis had greater prostatic oxidative stress/DNA damage, and prostate epithelial cells had undergone cell cycle arrest.Conclusions:These data suggest chronic intestinal inflammation is associated with an inflammatory-rich, pro-tumorigenic prostatic phenotype which may explain how gut inflammation fosters prostate cancer development in men with IBD.

Trop2 regulates prostate cancer growth and metastasis through distinct molecular mechanisms

Background Prostate cancer is the most common non-cutaneous cancer and second leading cause of cancer related deaths in men in the United States. The first line of treatment for men with advanced prostate cancer is androgen deprivation therapy. Although initial responses are observed, prostate cancer commonly relapses in its lethal metastatic form referred to as castration resistant prostate cancer (CRPC) with 1-2 years mean survival time. Neuroendocrine prostate cancer (NEPC) is highly aggressive, AR independent subtype and usually emerges post castration resistance. We recently identified that the cell surface receptor, Trop2, is a new driver of NEPC. Moreover, we demonstrated that Trop2 regulates prostate cancer growth and metastasis. In this study, we set out to delineate the molecular mechanisms through which Trop2 regulates prostate cancer growth and metastasis. Methods Proximity-dependent Biotin Identification (BioID) followed by mass spectrometry was performed to identify Trop2 interactome. Lentiviral infection was used to generate prostate cancer cell lines with over-expression of Trop2 and knock-down of NOTCH1, SLC4A7, PLEC, and OCLN shRNA to modulate gene expression levels.In vitro functional assays were performed includingcolony formation, and Matrigel drop 3D cell invasion assays. Results The Trop2 membrane interactome was identified utilizing Proximity-dependent Biotin Identification (BioID) in living cells and uncovered that Trop2-mediated prostate cancer growth and metastasis are orchestrated by distinct downstream pathways including Notch signaling (NOTCH1), control of intracellular pH (SLC4A7), exosome secretion (PLEC), and tight junctions (OCLN). Interaction of Trop2 binding partners including NOTCH1, SLC4A7, PLEC and OCLN with Trop2 were further validated by fluorescence resonance energy transfer (FRET) using confocal microscopy. Moreover, knocking down the Trop2 interacting partners in Trop2 over-expressing prostate cancer cells suppressed Trop2-driven growth and invasion ability. Conclusions In a previous study, we identified cell surface receptor, Trop2, as a novel driver of metastatic NEPC. Herein, our new findings reveal that Trop2 interacts with NOTCH1, SLC4A7, PLEC, and OCLN, which may highlight novel biological functions of Trop2 in prostate tumorigenesis and provide new understanding of the potential mechanism of neuroendocrine differentiation and metastasis to provide new therapeutic strategy for metastatic CRPC with neuroendocrine features.