Citations

DNA replication is a vulnerable cellular process, and its deregulation leads to genomic instability. Here, we demonstrate that chromobox protein homolog 3 (CBX3) binds replication protein A 32-kDa subunit (RPA2) and regulates RPA2 retention at stalled replication forks. CBX3 is recruited to stalled replication forks by RPA2 and inhibits ring finger and WD repeat domain 3 (RFWD3)–facilitated replication restart. Phosphorylation of CBX3 at serine-95 by casein kinase 2 (CK2) kinase augments cadherin 1 (CDH1)–mediated CBX3 degradation and RPA2 dynamics at stalled replication forks, which permits replication fork restart. Increased expression of CBX3 due to gene amplification or CK2 inhibitor treatment sensitizes prostate cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors while inducing replication stress and DNA damage. Our work reveals CBX3 as a key regulator of RPA2 function and DNA replication, suggesting that CBX3 could serve as an indicator for targeted therapy of cancer using PARP inhibitors.

Abstract Introduction: Porcine myeloperoxidase (pMPO) is a cationic enzyme secreted by granulocytic leukocytes capable of selectively binding cancer cells due to their negative surface charge. In the presence of hydrogen peroxide (H2O2) and sodium chloride (NaCl), MPO catalyzes the production of electronically excited singlet molecular oxygen, which exerts focused cytotoxic activity against cancer cells. Methods: Human bladder non-cancer (SV-HUC1) and cancer cells (T24 and 5637) were used to assess differential cytotoxic activity of pMPO. pMPO was combined with associated reagents, including H2O2, NaCl, and L-proline and glycine for 45 minutes to support the generation of singlet oxygen. MTS assay was used to assessed cell viability at day 3 post-treatment. Non-cancer and cancer cells were labeled with GFP and mCherry, respectively, via infection with lentivirus and cultured in tandem to allow for simultaneous treatment of both cell types. After the treatment, flow cytometry and microscopy were used to monitor the growth of the cells. Results: No cytotoxicity was noted when SV-HUC1 cells were cultured with individual reagents pMPO, NaCl, L-Proline, or Glycine, and limited cytotoxicity was observed with H2O2. Notably, cell viability was nearly maximally preserved for SV-HUC1 cells. The combination of pMPO with associated reagents resulted in killing of 5637 and T24 cells, with tumoricidal activity maintained across increasing tumor cell density, with cell viability preserved for SV-HUC1 cells. Within the cell co-culture system, simultaneous treatment of both cell types with the pMPO treatment resulted in selective killing of tumor cells and preservation of SV-HUC1 cells. Mechanistic studies confirm specificity of pMPO binding to tumor cells, and further, high levels of expression of gammaH2AX and phospho-ATM in 5637 and T24 cells following treatment. Conclusion: pMPO demonstrates selective and efficient killing of bladder cancer cells with minimal cytotoxicity against non-cancer urothelial cells. Given its reliance on direct surface contact with tumor cells for activity, pMPO may be developed as an intravesical therapy for treatment of non-muscle invasive bladder cancers. Acknowledgements: This work was partially supported by Mayo funds provided to Dr. Haojie Huang. Citation Format: Junnan Liu, Robert C. Allen, Jackson T. Stephens, Paras Shah, Haojie Huang. Investigation of anti-tumor activity of porcine myeloperoxidase for urothelial carcinoma of the bladder [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2029.

AbstractSenescent cells compromise tissue structure and function in older organisms. We recently identified senescent fibroadipogenic progenitors (FAPs) with activated chemokine signaling pathways in the skeletal muscle of old mice, and hypothesized these cells may contribute to the age‐associated accumulation of immune cells in skeletal muscle. In this study, through cell–cell communication analysis of skeletal muscle single‐cell RNA‐sequencing data, we identified unique interactions between senescent FAPs and macrophages, including those mediated by Ccl2 and Spp1. Using mouse primary FAPs in vitro, we verified increased expression of Ccl2 and Spp1 and secretion of their respective proteins in the context of both irradiation‐ and etoposide‐induced senescence. Compared to non‐senescent FAPs, the medium of senescent FAPs markedly increased the recruitment of macrophages in an in vitro migration assay, an effect that was mitigated by preincubation with antibodies to either CCL2 or osteopontin (encoded by Spp1). Further studies demonstrated that the secretome of senescent FAPs promotes polarization of macrophages toward an M2 subtype. These data suggest the unique secretome of senescent FAPs may compromise skeletal muscle homeostasis by recruiting and directing the behavior of macrophages.

Abstract Intraductal carcinoma of the prostate (IDC-P) is a lethal prostate cancer subtype that generally coexists with invasive high-grade prostate acinar adenocarcinoma (PAC) but exhibits distinct biological features compared with concomitant adenocarcinoma. In this study, we performed whole-exome, RNA, and DNA-methylation sequencing of IDC-P, concurrent invasive high-grade PAC lesions, and adjacent normal prostate tissues isolated from 22 radical prostatectomy specimens. Three evolutionary patterns of concurrent IDC-P and PAC were identified: early divergent, late divergent, and clonally distant. In contrast to those with a late divergent evolutionary pattern, tumors with clonally distant and early divergent evolutionary patterns showed higher genomic, epigenomic, transcriptional, and pathologic heterogeneity between IDC-P and PAC. Compared with coexisting PAC, IDC-P displayed increased expression of adverse prognosis–associated genes. Survival analysis based on an independent cohort of 505 patients with metastatic prostate cancer revealed that IDC-P carriers with lower risk International Society of Urological Pathology (ISUP) grade 1–4 adenocarcinoma displayed a castration-resistant free survival as poor as those with the highest risk ISUP grade 5 tumors that lacked concurrent IDC-P. Furthermore, IDC-P exhibited robust cell-cycle progression and androgen receptor activities, characterized by an enrichment of cellular proliferation–associated master regulators and genes involved in intratumoral androgen biosynthesis. Overall, this study provides a molecular groundwork for the aggressive behavior of IDC-P and could help identify potential strategies to improve treatment of IDC-P. Significance: The genomic, transcriptomic, and epigenomic characterization of concurrent intraductal carcinoma and adenocarcinoma of the prostate deepens the biological understanding of this lethal disease and provides a genetic basis for developing targeted therapies.

SPOP mutations drive opposing molecular and immune landscapes in prostate and endometrial cancers, suggesting tailored therapies

Abstract Androgen receptor (AR) inhibition by androgen deprivation and/or antiandrogen administration is the mainstay therapy for advanced prostate cancer. However, most prostate cancers ultimately become resistant to these therapies, indicating the importance of identifying mechanisms driving resistance to improve patient outcomes. Here we demonstrated that acute treatment with the antiandrogen enzalutamide (ENZ) decreased glutathione (GSH) production, increased lipid peroxidation, and induced ferroptosis in prostate cancer cells. Consistently, meta-analysis of transcriptomic data linked the androgen-AR axis to metabolism-related biological processes, including lipid metabolism. The cystine transporter gene SLC7A11 was a key AR target, and full-length AR (AR-FL) transactivated SLC7A11 transcription by directly occupying the SLC7A11 promoter and putative enhancer regions. AR variants (AR-V) preferentially bound the SLC7A11 enhancer and upregulated SLC7A11 expression, thereby conferring resistance to ferroptosis induced by ENZ treatment. However, this effect was abolished following downregulation of AR-Vs using the dual CBP/p300 and BET inhibitor NEO2734. These findings reveal ferroptosis induction as an anticancer mechanism of antiandrogens and SLC7A11 as a direct target gene of AR-FL and AR-Vs. AR-V-mediated SLC7A11 expression represents a mechanism coupling ferroptosis resistance to prostate cancer progression. Significance: Upregulation of SLC7A11 can be induced by androgen receptor variants to inhibit antiandrogen-induced prostate cancer cell ferroptosis and to drive castration resistance in prostate cancer.

Abstract Retinoblastoma (RB) protein can exert tumor suppressor functions even when it becomes phosphorylated. It is thus essential to understand how phosphorylated RB (p-RB) expression and function are regulated. Here, we demonstrated that RING finger domain protein TRIM28 bound and promoted ubiquitination and degradation of CDK4/6-phosphorylated RB protein. SETDB1, a known TRIM28 binding partner, protected p-RB from degradation through the binding of methylated RB by its Tudor domain independent of its methyltransferase activity. SETDB1 was found to be frequently overexpressed due to gene amplification and positively correlated with p-RB in prostate cancer patient specimens. Inhibition of SETDB1 expression using a gene-specific antisense oligonucleotide (ASO) reduced tumor growth but accelerated RB protein degradation, limiting the therapeutic efficacy. However, coadministration of the CDK4/6 inhibitor palbociclib blocked ASO-induced RB degradation and resulted in a much greater cancer-inhibitory effect than each inhibitor alone both in vitro and in vivo. This study identified CDK4/6-dependent, TRIM28-mediated proteasomal degradation as a mechanism of RB inactivation and reveals SETDB1 as a key inhibitor of this process. Our findings suggest that combined targeting of SETDB1 and CDK4/6 represents a viable approach for the treatment of cancers with SETDB1 gene amplification or overexpression. Significance: The identification of a role for TRIM28 and SETDB1 in regulating CDK4/6-phosphorylated RB stability uncovers a combination strategy using CDK4/6 and SETDB1 inhibition to decrease RB degradation and inhibit cancer growth.

Abstract Speckle-type POZ protein (SPOP) is a substrate-binding adaptor of the CULLIN3/RING-box1 E3 ubiquitin ligase complex. SPOP is frequently mutated in prostate and endometrial cancers, whereas it is overexpressed in renal cell carcinoma (RCC). SPOP can mediate both degradable and nondegradable polyubiquitination of a number of substrates with diverse biological functions such as androgen receptor (AR), SRC-3, TRIM24, BRD4, PD-L1, 53BP1, GLP/G9a, c-Myc, SENP7, among others. Cancer-associated SPOP mutants often impair SPOP binding and polyubiquitination of its substrates to influence various cancer-relevant pathways, which include androgen/AR signaling, DNA repair and methylation, cellular stress surveillance, cancer metabolism, and immunity. Although SPOP is recognized as a tumor suppressor in prostate and endometrial cancers, it acts like an oncoprotein in RCC. This review provides an overview of the recent progress in understanding of the upstream regulators of SPOP and its downstream targets, highlights the significant impact of SPOP mutations and overexpression on cancer pathogenesis, and discusses the potential of targeting SPOP for cancer treatment.

Androgen receptor (AR) messenger RNA (mRNA) alternative splicing variants (AR-Vs) are implicated in castration-resistant progression of prostate cancer (PCa), although the molecular mechanism underlying the genesis of AR-Vs remains poorly understood. The CDK12 gene is often deleted or mutated in PCa and CDK12 deficiency is known to cause homologous recombination repair gene alteration or BRCAness via alternative polyadenylation (APA). Here, we demonstrate that pharmacological inhibition or genetic inactivation of CDK12 induces AR gene intronic (intron 3) polyadenylation (IPA) usage, AR-V expression, and PCa cell resistance to the antiandrogen enzalutamide (ENZ). We further show that AR binds to the CCNK gene promoter and up-regulates CYCLIN K expression. In contrast, ENZ decreases AR occupancy at the CCNK gene promoter and suppresses CYCLIN K expression. Similar to the effect of the CDK12 inhibitor, CYCLIN K degrader or ENZ treatment promotes AR gene IPA usage, AR-V expression, and ENZ-resistant growth of PCa cells. Importantly, we show that targeting BRCAness induced by CYCLIN K down-regulation with the PARP inhibitor overcomes ENZ resistance. Our findings identify CYCLIN K down-regulation as a key driver of IPA usage, hormonal therapy–induced AR-V expression, and castration resistance in PCa. These results suggest that hormonal therapy–induced AR-V expression and therapy resistance are vulnerable to PARP inhibitor treatment.

Abstract NRG-1 signals through epidermal growth factor receptor (EGFR) family members. Despite promising preclinical data, HER2 blockade in prostate cancer has failed to improve outcomes in clinical trials.1-4 We have discovered a novel complex systemic role of NRG-1 that drives sensitivity and resistance to androgen deprivation therapy (ADT) in castration-sensitive (CSPC) and castration resistant prostate cancer (CRPC), respectively. Significantly, sensitivity to ADT can be restored in CRPC by manipulating NRG-1 signaling. Our data posit NRG-1 as a systemic “switch” in prostate cancer. Plasma NRG-1 is elevated in our cohort of patients with metastatic CRPC (mean 4.9ng/ml) versus healthy controls (mean 3.2ng/ml, p<0.0001) or metastatic CSPC (mean 3.4ng/ml, p<0.0001). Paradoxically, high systemic NRG-1 predicted superior OS and PFS in CSPC but inferior clinical outcomes in CRPC. Consistently, opposite cell survival between LNCaP (CSPC) vs Du145 & PC3 (CRPC) was observed with recombinant human NRG-1 and enzalutamide (ENZ). ENZ combined with increasing NRG-1 concentration induced LNCaP cell death in an additive manner, but NRG-1 rescued Du145 & PC3 from ENZ-induced death in a dose-dependent manner. We next hypothesized that differential NRG-1 downstream signaling is mediated by engagement with distinct EGFR receptor dimers. While HER2 expression is similar, LNCaP express higher HER3 than Du145 & PC3. High ratio of HER2 to HER3 in Du145 & PC3 mirrors clinical reports of HER2 upregulation in CRPC5,6 and also predicts patient survival in the Stand Up to Cancer cohort.7 Significantly, we recapitulated the CRPC phenotype in LNCaP by inducing HER2 overexpression, HER3 suppression, and chemically-mediated HER2/HER3 dimerization. Conversely, HER2 knockdown sensitized Du145 cells to ENZ. Next, we queried composite single-cell whole human8 and CRPC9 transcriptomes for NRG-1 expression and discovered that the source of NRG-1 is the patients’ own mesenchymal stem cells (MSCs). Bone-derived MSC outgrowth cells produced copious NRG-1, which was increased substantially by coincubation with prostate cancer cell supernatants. Importantly, we were able to manipulate MSCs to reduce their NRG-1 production as a possible clinical translation. Similarly, patients treated with these compounds showed significant reduction in plasma NRG-1 levels after treatment. Additional clinical studies are pending. Together, our observations confirm that NRG-1 has differential signaling: HER3 homodimerization leads to prostate cancer cell death, whereas heterodimerization of HER3 with HER2 leads to prostate cancer cell survival via ADT resistance in a plasma NRG-1 dependent manner. Consequently, HER2 and HER3 receptor availability determine the molecular switch of NRG-1 in prostate cancer. Therefore, NRG-1 can be both a prognostic marker and therapeutic target in CRPC, and multiple clinical trials are pending. Citation Format: Jacob Orme, Roman Thaler, Dejie Wang, Siven Chinniah, Jacob Hirdler, Fernando Quevedo, Lance Pagliaro, Kwon Eugene, Alan Bryce, Brian Costello, Haojie Huang, Sean Park. Mesenchymal stem cell-derived systemic NRG-1 mediates a targetable molecular switch in metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 397.