PARP inhibition and pharmacological ascorbate demonstrate synergy in castration-resistant prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer death for men in the United States. While organ-confined disease has reasonable expectation of cure, metastatic PCa is universally fatal upon recurrence during hormone therapy, a stage termed castration-resistant prostate cancer (CRPC). Until such time as molecularly defined subtypes can be identified and targeted using precision medicine, it is necessary to investigate new therapies that may apply to the CRPC population as a whole. The administration of ascorbate, more commonly known as ascorbic acid or Vitamin C, has proved lethal to and highly selective for a variety of cancer cell types. There are several mechanisms currently under investigation to explain how ascorbate exerts anti-cancer effects. A simplified model depicts ascorbate as a pro-drug for reactive oxygen species (ROS), which accumulate intracellularly and generate DNA damage. It was therefore hypothesized that poly(ADP-ribose) polymerase (PARP) inhibitors, by inhibiting DNA damage repair, would augment the toxicity of ascorbate. Results: Two distinct CRPC models were found to be sensitive to physiologically relevant doses of ascorbate. Moreover, additional studies indicate that ascorbate inhibits CRPC growth in vitro via multiple mechanisms including disruption of cellular energy dynamics and accumulation of DNA damage. Combination studies were performed in CRPC models with ascorbate in conjunction with escalating doses of three different PARP inhibitors (niraparib, olaparib, and talazoparib). The addition of ascorbate augmented the toxicity of all three PARP inhibitors and proved synergistic with olaparib in both CRPC models. Finally, the combination of olaparib and ascorbate was tested in vivo in both castrated and non-castrated models. In both cohorts, the combination treatment significantly delayed tumor growth compared to monotherapy or untreated control. Conclusions: These data indicate that pharmacological ascorbate is an effective monotherapy at physiological concentrations and kills CRPC cells. Ascorbate-induced tumor cell death was associated with disruption of cellular energy dynamics and accumulation of DNA damage. The addition of PARP inhibition increased the extent of DNA damage and proved effective at slowing CRPC growth both in vitro and in vivo. These findings nominate ascorbate and PARPi as a novel therapeutic regimen that has the potential to improve CRPC patient outcomes.

A Novel Role for DNA-PK in Metabolism by Regulating Glycolysis in Castration-Resistant Prostate Cancer.

PURPOSE: DNA-dependent protein kinase catalytic subunit (DNA-PKcs, herein referred as DNA-PK) is a multifunctional kinase of high cancer relevance. DNA-PK is deregulated in multiple tumor types, including prostate cancer, and is associated with poor outcomes. DNA-PK was previously nominated as a therapeutic target and DNA-PK inhibitors are currently undergoing clinical investigation. Although DNA-PK is well studied in DNA repair and transcriptional regulation, much remains to be understood about the way by which DNA-PK drives aggressive disease phenotypes. EXPERIMENTAL DESIGN: Here, unbiased proteomic and metabolomic approaches in clinically relevant tumor models uncovered a novel role of DNA-PK in metabolic regulation of cancer progression. DNA-PK regulation of metabolism was interrogated using pharmacologic and genetic perturbation using in vitro cell models, in vivo xenografts, and ex vivo in patient-derived explants (PDE). RESULTS: Key findings reveal: (i) the first-in-field DNA-PK protein interactome; (ii) numerous DNA-PK novel partners involved in glycolysis; (iii) DNA-PK interacts with, phosphorylates (in vitro), and increases the enzymatic activity of glycolytic enzymes ALDOA and PKM2; (iv) DNA-PK drives synthesis of glucose-derived pyruvate and lactate; (v) DNA-PK regulates glycolysis in vitro, in vivo, and ex vivo; and (vi) combination of DNA-PK inhibitor with glycolytic inhibitor 2-deoxyglucose leads to additive anti-proliferative effects in aggressive disease. CONCLUSIONS: Findings herein unveil novel DNA-PK partners, substrates, and function in prostate cancer. DNA-PK impacts glycolysis through direct interaction with glycolytic enzymes and modulation of enzymatic activity. These events support energy production that may contribute to generation and/or maintenance of DNA-PK-mediated aggressive disease phenotypes.

Novel Oncogenic Transcription Factor Cooperation in RB-Deficient Cancer.

The retinoblastoma tumor suppressor (RB) is a critical regulator of E2F-dependent transcription, controlling a multitude of protumorigenic networks including but not limited to cell-cycle control. Here, genome-wide assessment of E2F1 function after RB loss in isogenic models of prostate cancer revealed unexpected repositioning and cooperation with oncogenic transcription factors, including the major driver of disease progression, the androgen receptor (AR). Further investigation revealed that observed AR/E2F1 cooperation elicited novel transcriptional networks that promote cancer phenotypes, especially as related to evasion of cell death. These observations were reflected in assessment of human disease, indicating the clinical relevance of the AR/E2F1 cooperome in prostate cancer. Together, these studies reveal new mechanisms by which RB loss induces cancer progression and highlight the importance of understanding the targets of E2F1 function. SIGNIFICANCE: This study identifies that RB loss in prostate cancer drives cooperation between AR and E2F1 as coregulators of transcription, which is linked to the progression of advanced disease.

Relevance of pRB Loss in Human Malignancies.

The retinoblastoma tumor suppressor protein (pRB) is a known regulator of cell-cycle control; however, recent studies identified critical functions for pRB in regulating cancer-associated gene networks that influence the DNA damage response, apoptosis, and cell metabolism. Understanding the impact of these pRB functions on cancer development and progression in the clinical setting will be essential, given the prevalence of pRB loss of function across disease types. Moreover, the current state of evidence supports the concept that pRB loss results in pleiotropic effects distinct from tumor proliferation. Here, the implications of pRB loss (and resultant pathway deregulation) on disease progression and therapeutic response will be reviewed, based on clinical observation. Developing a better understanding of the pRB-regulated pathways that underpin the aggressive features of pRB-deficient tumors will be essential for further developing pRB as a biomarker of disease progression and for stratifying pRB-deficient tumors into more effective treatment regimens.

Mutant p53 elicits context-dependent pro-tumorigenic phenotypes.

The tumor suppressor gene TP53 is the most frequently mutated gene in numerous cancer types, including prostate cancer (PCa). Specifically, missense mutations in TP53 are selectively enriched in PCa, and cluster to particular "hot spots" in the p53 DNA binding domain with mutation at the R273 residue occurring most frequently. While this residue is similarly mutated to R273C-p53 or R273H-p53 in all cancer types examined, in PCa selective enrichment of R273C-p53 is observed. Importantly, examination of clinical datasets indicated that TP53 heterozygosity can either be maintained or loss of heterozygosity (LOH) occurs. Thus, to mimic tumor-associated mutant p53, R273C-p53 and R273H-p53 isogenic PCa models were developed in the presence or absence of wild-type p53. In the absence of wild-type p53, both R273C-p53 and R273H-p53 exhibited similar loss of DNA binding, transcriptional profiles, and loss of canonical tumor suppressor functions associated with wild-type p53. In the presence of wild-type p53 expression, both R273C-p53 and R273H-p53 supported canonical p53 target gene expression yet elicited distinct cistromic and transcriptional profiles when compared to each other. Moreover, heterozygous modeling of R273C-p53 or R273H-p53 expression resulted in distinct phenotypic outcomes in vitro and in vivo. Thus, mutant p53 acts in a context-dependent manner to elicit pro-tumorigenic transcriptional profiles, providing critical insight into mutant p53-mediated prostate cancer progression.

RB/E2F1 as a Master Regulator of Cancer Cell Metabolism in Advanced Disease.

Loss of the retinoblastoma (RB) tumor suppressor protein is a critical step in reprogramming biological networks that drive cancer progression, although mechanistic insight has been largely limited to the impact of RB loss on cell-cycle regulation. Here, isogenic modeling of RB loss identified disease stage-specific rewiring of E2F1 function, providing the first-in-field mapping of the E2F1 cistrome and transcriptome after RB loss across disease progression. Biochemical and functional assessment using both in vitro and in vivo models identified an unexpected, prominent role for E2F1 in regulation of redox metabolism after RB loss, driving an increase in the synthesis of the antioxidant glutathione, specific to advanced disease. These E2F1-dependent events resulted in protection from reactive oxygen species in response to therapeutic intervention. On balance, these findings reveal novel pathways through which RB loss promotes cancer progression and highlight potentially new nodes of intervention for treating RB-deficient cancers. SIGNIFICANCE: This study identifies stage-specific consequences of RB loss across cancer progression that have a direct impact on tumor response to clinically utilized therapeutics. The study herein is the first to investigate the effect of RB loss on global metabolic regulation and link RB/E2F1 to redox control in multiple advanced diseases.This article is highlighted in the In This Issue feature, p. 2113.

Differential expression of αVß3 and αVß6 integrins in prostate cancer progression.

Neuroendocrine prostate cancer (NEPrCa) arises de novo or after accumulation of genomic alterations in pre-existing adenocarcinoma tumors in response to androgen deprivation therapies. We have provided evidence that small extracellular vesicles released by PrCa cells and containing the αVß3 integrin promote neuroendocrine differentiation of PrCa in vivo and in vitro. Here, we examined αVß3 integrin expression in three murine models carrying a deletion of PTEN (SKO), PTEN and RB1 (DKO), or PTEN, RB1 and TRP53 (TKO) genes in the prostatic epithelium; of these three models, the DKO and TKO tumors develop NEPrCa with a gene signature comparable to those of human NEPrCa. Immunostaining analysis of SKO, DKO and TKO tumors shows that αVß3 integrin expression is increased in DKO and TKO primary tumors and metastatic lesions, but absent in SKO primary tumors. On the other hand, SKO tumors show higher levels of a different αV integrin, αVß6, as compared to DKO and TKO tumors. These results are confirmed by RNA-sequencing analysis. Moreover, TRAMP mice, which carry NEPrCa and adenocarcinoma of the prostate, also have increased levels of αVß3 in their NEPrCa primary tumors. In contrast, the αVß6 integrin is only detectable in the adenocarcinoma areas. Finally, analysis of 42 LuCaP patient-derived xenografts and primary adenocarcinoma samples shows a positive correlation between αVß3, but not αVß6, and the neuronal marker synaptophysin; it also demonstrates that αVß3 is absent in prostatic adenocarcinomas. In summary, we demonstrate that αVß3 integrin is upregulated in NEPrCa primary and metastatic lesions; in contrast, the αVß6 integrin is confined to adenocarcinoma of the prostate. Our findings suggest that the αVß3 integrin, but not αVß6, may promote a shift in lineage plasticity towards a NE phenotype and might serve as an informative biomarker for the early detection of NE differentiation in prostate cancer.

The circadian cryptochrome, CRY1, is a pro-tumorigenic factor that rhythmically modulates DNA repair.

Mechanisms regulating DNA repair processes remain incompletely defined. Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target.

Double Trouble: Concomitant RB1 and BRCA2 Depletion Evokes Aggressive Phenotypes.

Coordinate single- or two copy loss of the BRCA2/RB1 tumor suppressor genes, which reside in close chromosomal proximity, were found to be associated with aggressive prostate cancer and therapeutic resistance. Modeling these events and analyses of human cancers suggest that dual depletion of BRCA2/RB1 may represent a distinct subtype of disease.See related article by Chakraborty et al., p. 2047.

USP22 Functions as an Oncogenic Driver in Prostate Cancer by Regulating Cell Proliferation and DNA Repair.

Emerging evidence indicates the deubiquitinase USP22 regulates transcriptional activation and modification of target substrates to promote pro-oncogenic phenotypes. Here, in vivo characterization of tumor-associated USP22 upregulation and unbiased interrogation of USP22-regulated functions in vitro demonstrated critical roles for USP22 in prostate cancer. Specifically, clinical datasets validated that USP22 expression is elevated in prostate cancer, and a novel murine model demonstrated a hyperproliferative phenotype with prostate-specific USP22 overexpression. Accordingly, upon overexpression or depletion of USP22, enrichment of cell-cycle and DNA repair pathways was observed in the USP22-sensitive transcriptome and ubiquitylome using prostate cancer models of clinical relevance. Depletion of USP22 sensitized cells to genotoxic insult, and the role of USP22 in response to genotoxic insult was further confirmed using mouse adult fibroblasts from the novel murine model of USP22 expression. As it was hypothesized that USP22 deubiquitylates target substrates to promote protumorigenic phenotypes, analysis of the USP22-sensitive ubiquitylome identified the nucleotide excision repair protein, XPC, as a critical mediator of the USP22-mediated response to genotoxic insult. Thus, XPC undergoes deubiquitylation as a result of USP22 function and promotes USP22-mediated survival to DNA damage. Combined, these findings reveal unexpected functions of USP22 as a driver of protumorigenic phenotypes and have significant implications for the role of USP22 in therapeutic outcomes. SIGNIFICANCE: The studies herein present a novel mouse model of tumor-associated USP22 overexpression and implicate USP22 in modulation of cellular survival and DNA repair, in part through regulation of XPC.

Pleiotropic Impact of DNA-PK in Cancer and Implications for Therapeutic Strategies.

PURPOSE: DNA-dependent protein kinase catalytic subunit (DNA-PK) is a pleiotropic kinase involved in DNA repair and transcriptional regulation. DNA-PK is deregulated in selected cancer types and is strongly associated with poor outcome. The underlying mechanisms by which DNA-PK promotes aggressive tumor phenotypes are not well understood. Here, unbiased molecular investigation in clinically relevant tumor models reveals novel functions of DNA-PK in cancer.Experimental Design: DNA-PK function was modulated using both genetic and pharmacologic methods in a series of in vitro models, in vivo xenografts, and patient-derived explants (PDE), and the impact on the downstream signaling and cellular cancer phenotypes was discerned. Data obtained were used to develop novel strategies for combinatorial targeting of DNA-PK and hormone signaling pathways. RESULTS: Key findings reveal that (i) DNA-PK regulates tumor cell proliferation; (ii) pharmacologic targeting of DNA-PK suppresses tumor growth both in vitro, in vivo, and ex vivo; (iii) DNA-PK transcriptionally regulates the known DNA-PK-mediated functions as well as novel cancer-related pathways that promote tumor growth; (iv) dual targeting of DNA-PK/TOR kinase (TORK) transcriptionally upregulates androgen signaling, which can be mitigated using the androgen receptor (AR) antagonist enzalutamide; (v) cotargeting AR and DNA-PK/TORK leads to the expansion of antitumor effects, uncovering the modulation of novel, highly relevant protumorigenic cancer pathways; and (viii) cotargeting DNA-PK/TORK and AR has cooperative growth inhibitory effects in vitro and in vivo. CONCLUSIONS: These findings uncovered novel DNA-PK transcriptional regulatory functions and led to the development of a combinatorial therapeutic strategy for patients with advanced prostate cancer, currently being tested in the clinical setting.

PARP-1 regulates DNA repair factor availability.

PARP-1 holds major functions on chromatin, DNA damage repair and transcriptional regulation, both of which are relevant in the context of cancer. Here, unbiased transcriptional profiling revealed the downstream transcriptional profile of PARP-1 enzymatic activity. Further investigation of the PARP-1-regulated transcriptome and secondary strategies for assessing PARP-1 activity in patient tissues revealed that PARP-1 activity was unexpectedly enriched as a function of disease progression and was associated with poor outcome independent of DNA double-strand breaks, suggesting that enhanced PARP-1 activity may promote aggressive phenotypes. Mechanistic investigation revealed that active PARP-1 served to enhance E2F1 transcription factor activity, and specifically promoted E2F1-mediated induction of DNA repair factors involved in homologous recombination (HR). Conversely, PARP-1 inhibition reduced HR factor availability and thus acted to induce or enhance "BRCA-ness". These observations bring new understanding of PARP-1 function in cancer and have significant ramifications on predicting PARP-1 inhibitor function in the clinical setting.

Differential impact of RB status on E2F1 reprogramming in human cancer.

The tumor suppressor protein retinoblastoma (RB) is mechanistically linked to suppression of transcription factor E2F1-mediated cell cycle regulation. For multiple tumor types, loss of RB function is associated with poor clinical outcome. RB action is abrogated either by direct depletion or through inactivation of RB function; however, the basis for this selectivity is unknown. Here, analysis of tumor samples and cell-free DNA from patients with advanced prostate cancer showed that direct RB loss was the preferred pathway of disruption in human disease. While RB loss was associated with lethal disease, RB-deficient tumors had no proliferative advantage and exhibited downstream effects distinct from cell cycle control. Mechanistically, RB loss led to E2F1 cistrome expansion and different binding specificity, alterations distinct from those observed after functional RB inactivation. Additionally, identification of protumorigenic transcriptional networks specific to RB loss that were validated in clinical samples demonstrated the ability of RB loss to differentially reprogram E2F1 in human cancers. Together, these findings not only identify tumor-suppressive functions of RB that are distinct from cell cycle control, but also demonstrate that the molecular consequence of RB loss is distinct from RB inactivation. Thus, these studies provide insight into how RB loss promotes disease progression, and identify new nodes for therapeutic intervention.