Huang, Haojie

Abstract CDK12 is frequently deleted and/or mutated in human cancers, and CDK12 inactivation is associated with aggressive phenotypes, including the tendency to metastasis. In this study, we showed that CDK12 deficiency induced intronic polyadenylation (IPA) usage in BRD4 to upregulate expression of the bromodomain-containing protein 4 (BRD4) short isoform (BRD4S). BRD4S demonstrated an enhanced capacity to form phase separation despite lacking the intrinsically disordered region located in the C-terminal of full-length BRD4. Oligomerization of BRD4S mediated phase separation through a base-interacting structural domain. CUT&Tag and RNA sequencing analyses revealed that BRD4S preferentially bound to the promoters of TGFβ signaling genes, including TGFB2 and LTBP1, driving their expression. Consistently, BRD4S drove cancer cell dissemination in vitro and metastasis in vivo, which could be blocked by BET or TGFβ signaling pathway inhibitors. Overall, this study identifies IPA usage at the BRD4 gene locus induced by CDK12 deficiency as a mechanism responsible for BRD4S genesis, which represents an important mechanism driving cancer metastasis and a potential target for therapeutic intervention in CDK12-deficient cancers. Significance: The CDK12–BRD4S–TGFβ signaling axis represents a key regulatory module that promotes metastatic progression and can be targeted by BET or TGFβ pathway inhibitors for treating CDK12-deficient cancers.

Prostate cancer (PCa) progression is driven by intricate molecular mechanisms involving dysregulated signaling networks and posttranslational modifications of key regulatory proteins. In this study, we identify a novel oncogenic pathway wherein cyclin-dependent kinase 12 (CDK12) physically interacts with and phosphorylates forkhead box A1 (FOXA1) at serine 234 (S234). Phosphorylation at this residue markedly enhances FOXA1 transcriptional activity, leading to up-regulation of downstream targets including murine double minute 2 (MDM2), a critical negative regulator of the p53 tumor suppressor. Mechanistically, this CDK12–FOXA1–MDM2 axis destabilizes p53, attenuates apoptotic signaling, and promotes PCa cell survival and proliferation. Therapeutic targeting of CDK12 using the small-molecule inhibitor THZ531 or RNA interference effectively abrogates FOXA1 phosphorylation, restores p53 stability, reactivates apoptotic pathways, and suppresses tumor growth. Notably, the identification of S234 as a functional phosphorylation site in FOXA1 reveals a previously uncharacterized posttranslational regulatory mechanism in PCa biology. These findings establish the CDK12–FOXA1–MDM2 axis as a pivotal driver of PCa progression and underscore the therapeutic potential of targeting FOXA1 phosphorylation to restore tumor suppressor function and induce apoptosis in PCa. Our work provides a mechanistic framework for developing precision therapies aimed at disrupting this oncogenic cascade in PCa.

Abstract The roles of nucleoplasm-residing nucleoporins (NUP) in solid tumors, including prostate cancer, remain unknown. In this study, we reveal the clinical significance and mechanistic role of the off-pore NUP, soluble POM121 (sPOM121), as a crucial transcriptional regulator that enhances the aggressiveness of metastatic prostate cancer. Using orthogonal methodologies in human samples, sPOM121 was identified as the predominantly expressed nucleoplasmic NUP in prostate cancer. Unbiased proteomic and epigenomic studies demonstrate that sPOM121, through its C-terminus, interacts with the chromatin remodeler SMARCA5 at gene promoter sites and localizes at nuclear condensates, reprogramming gene expression. Indeed, sPOM121 regulates a distinct oncogenic gene network, including β-catenin, leading to prostate cancer progression and immune evasion. Importantly, targeting the sPOM121/β-catenin axis in patient-derived preclinical and syngeneic mouse models halts prostate cancer aggressiveness and enhances antitumor immunity. Taken together, these findings reveal previously unknown actionable reprogramming functions of off-pore NUPs in solid tumors. Significance: This study uncovers how oncogenic signaling programs are transcriptionally heightened by the NUP sPOM121 in metastatic prostate cancer. Localization of sPOM121 at active transcriptional nuclear condensates propels disease progression and immune evasion, offering novel anticancer therapeutic opportunities.

CDK12 primarily functions as a transcription regulatory cyclin-dependent kinase (CDK) that controls mRNA elongation, splicing, and polyadenylation. The CDK12 gene is implicated in human cancers since it is frequently mutated and/or deleted in prostate and ovarian cancer but paradoxically amplified in breast cancer. Here, we demonstrate that CDK12 promotes serine-933 phosphorylation of DNA2, a nuclease/helicase critical for replication fork stress regulation, and the phosphorylation subsequently facilitates DNA2 polyubiquitination and degradation mediated by the APC/C CDC20 E3 ubiquitin ligase. CDK12 inactivation induces but amplification suppresses genome-wide expression of interferon response and antigen processing and presentation machinery genes in ovarian and breast cancer cells, respectively. Besides causing aberrant DNA2 stabilization, replication stress, genomic instability, and cytosolic double-stranded DNA (dsDNA) accumulation, CDK12 loss also triggers cGAS–STING activation and innate immune response, which can be reversed by forced expression of replication protein A (RPA) subunits or DNA2 depletion. Our findings identify DNA2 as a phosphorylation substrate of CDK12, connecting CDK12 to cell cycle regulation. These data also reveal DNA2 protein destruction as a critical mechanism that dictates genomic instability, cGAS–STING signaling activation, and innate immune response in CDK12-deregulated cancers.

Abstract Approximately 50% of prostate cancer (PCa) patients harbor fusions involving the TMPRSS2 and ERG genes. Despite this, tailored therapies targeting the fused gene, tERG , remain undeveloped. Our study analyzed biopsy samples from two clinical trials assessing the efficacies of androgen receptor (AR) signaling inhibitors (ARSIs). The results revealed that tERG promotes resistance to ARSIs and is associated with elevated levels of the glucocorticoid receptor (GR). Subsequent assays showed that GR directly interacts with tERG, alleviates allosteric autoinhibition and prevents chemotherapy-induced tERG degradation. In PCa models, either inhibiting GR or lowering cortisol levels suppressed tumor growth in tERG-positive models, but not in fusion-negative models. In addition, patient-derived fusion-positive xenografts displayed enhanced sensitivity to combined GR and AR inhibitors. Collectively, these findings highlight TMPRSS2-ERG as a new biomarker and propose that simultaneous inhibition of GR and AR may specifically benefit tERG -positine patients. However, GR stimulatory corticosteroid therapies may not be advisable for this patient subgroup.

(1) Background: Prostate cancer treatment efficacy is significantly influenced by androgen receptor (AR) signaling pathways. SLC22A3, a membrane transporter, has been linked to SNP rs9364554 risk loci for drug efficacy in prostate cancer. (2) Methods: We examined the location of SNP rs9364554 in the genome and utilized TCGA and other publicly available datasets to analyze the association of this SNP with SLC22A3 transcription levels. We verified onco-mining findings in prostate cancer cell lines using quantitative PCR and Western blots. Additionally, we employed electrophoretic mobility shift assay (EMSA) to detect the binding affinity of transcription factors to this SNP. The ChIP-Seq was used to analyze the enrichment of H3K27ac on the SLC22A3 promoter. (3) Results: In this study, we revealed that SNP rs9364554 resides in the SLC22A3 gene and affects its transcription. The downregulation of SLC22A3 is associated with drug resistance. More importantly, we found that this SNP has different binding affinities with transcription factors, specifically FOXA1 and AR, which significantly affects their regulation of SLC22A3 transcription. (4) Conclusions: Our findings highlight the potential of using this SNP as a biomarker for predicting chemotherapeutic outcomes and uncover possible mechanisms underlying drug resistance in advanced prostate cancers. More importantly, it provides a clinical foundation for targeting FOXA1 to enhance drug efficacy in prostate cancer patients.

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.