Combination of PI3K/Akt Pathway Inhibition and Plk1 Depletion Can Enhance Chemosensitivity to Gemcitabine in Pancreatic Carcinoma

The prognosis of pancreatic cancer (PC) remains pessimistic because of the difficulty in early diagnosis as well as the little advance in chemotherapy. Although being the first-line chemotherapy drug for PC at present, gemcitabine still has some disadvantages, such as low drug sensitivity and significant side effects. Thus, how to further improve the sensitivity of PC cells to gemcitabine is still a difficult subject in the field of pancreatic cancer-treatment. Polo-like kinase 1 (Plk1) is closely related to poor outcome in many malignant tumors and its high expression is linked to chemoresistance in PC. As a downstream gene activated by PI3K/Akt signal pathway, we assumed that the targeted depletion of Plk1 could contribute to the chemosensitization induced by synergistic drug interaction of PI3K inhibitor LY294002 together with gemcitabine. To analyze effect of Plk1 in chemotherapy, we constructed two recombinant adenoviral vectors which carry enhanced green fluorescent protein (rAd-EGFP) and Plk1-shRNA (rAd-shPlk1), respectively. Both inhibition of PI3K/Akt signal pathway through PI3K inhibitor LY294002 and targeted depletion of Plk1 via recombinant adenoviral shRNA can cause chemosensitization, and the targeted depletion of Plk1 can enhance the chemosensitization of LY294002. Thus, the gene therapy like targeted depletion of Plk1 may create new perspectives for chemosensitization of PC.     

Cold Shock Domain Containing E1 (CSDE1) Protein is Overexpressed and Can be Targeted to Inhibit Invasiveness in Pancreatic Cancer Cells

Pancreatic cancer has a dismal prognosis and to date there are no targeted therapies for this malignancy. Using shotgun proteomics, the mRNA binding protein cold shock domain containing E1 (CSDE1), also called upstream‐of‐N‐Ras, is detected in pancreatic cancer cell lines but not in normal pancreatic epithelial cells. The expression of CSDE1 in pancreatic cancer cells is confirmed by Western blotting and immunohistochemistry of human pancreatic tumors. In vitro functional assays show that siRNA downregulation of CSDE1 or gene knockout using CRISPR‐Cas9 significantly reduce the invasiveness of pancreatic cancer cells. Together, this study reveals that CSDE1 is overexpressed in pancreatic cancer and is a potential therapeutic target to inhibit pancreatic cancer cell invasion.     

Involvement of p38 mitogen-activated protein kinase in gemcitabine-induced apoptosis in human pancreatic cancer cells

In this study, we investigated the involvement of Akt and members of the mitogen-activated protein kinase (MAPK) superfamily, including ERK, JNK, and p38 MAPK, in gemcitabine-induced cytotoxicity in human pancreatic cancer cells. We found that gemcitabine induces apoptosis in PK-1 and PCI-43 human pancreatic cancer cell lines. Gemcitabine specifically activated p38 MAPK in a dose- and time-dependent manner. A selective p38 MAPK inhibitor, SB203580, significantly inhibited gemcitabine-induced apoptosis in both cell lines, suggesting that phosphorylation of p38 MAPK may play a key role in gemcitabine-induced apoptosis in pancreatic cancer cells. A selective JNK inhibitor, SP600125, failed to inhibit gemcitabine-induced apoptosis in both cell lines. MKK3/6, an upstream activator of p38 MAPK, was phosphorylated by gemcitabine, indicating that the MKK3/6-p38 MAPK signaling pathway is indeed involved in gemcitabine-induced apoptosis. Furthermore, gemcitabine-induced cleavage of the caspase substrate poly(ADP-ribose) polymerase was inhibited by pretreatment with SB203580, suggesting that activation of p38 MAPK by gemcitabine induces apoptosis through caspase signaling. These results together suggest that gemcitabine-induced apoptosis in human pancreatic cancer cells is mediated by the MKK3/6-p38 MAPK-caspase signaling pathway. Further, these results lead us to suggest that p38 MAPK should be investigated as a novel molecular target for human pancreatic cancer therapies.     

Cotargeting Polo-Like Kinase 1 and the Wnt/β-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of β-catenin by enhancing binding between glycogen synthase kinase 3β (GSK3β) and β-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.     

Fate of primary cells at the G1/S boundary after polo-like kinase 1 inhibition by SBE13

Human polo-like kinase 1 (Plk1), a key regulator of mitosis, is over-expressed in various human tumors. It is a negative prognostic factor for cancer patients and a measure for the aggressiveness of a tumor. Thus, targeting Plk1 might be a promising approach for cancer therapy. Plk1 inhibitors represent attractive tools for cancer research and for the mechanistic investigation of checkpoint control. Here, we show the impact of Plk1 inhibition on cell cycle regulation in primary cells. After treatment with SBE13 the G1/S checkpoint was intact, indicated by reduced pRb, resulting in slower cell cycle progression but overall cell proliferation was not significantly impaired. Thus, we provide strong evidence that SBE13 leaves checkpoint control in primary cells unaffected making it a remarkable future anti-cancer therapeutic.     

Dissociation of gemcitabine chemosensitization by CHK1 inhibition from cell cycle checkpoint abrogation and aberrant mitotic entry

In order to determine the relative contribution of checkpoint abrogation and subsequent aberrant mitotic entry to gemcitabine chemosensitization by CHK1 inhibition, we established a model utilizing the CDK inhibitors roscovitine or purvalanol A to re-establish cell cycle arrest and prevent aberrant mitotic entry in pancreatic cancer cells treated with gemcitabine and the CHK inhibitor AZD7762. In this study, we report that the extent of aberrant mitotic entry, as determined by flow cytometry for the mitotic marker phospho-Histone H3 (Ser10), did not reflect the relative sensitivities of pancreatic cancer cell lines to gemcitabine chemosensitization by AZD7762. In addition, re-establishing gemcitabine-induced cell cycle arrest either pharmacologically, with roscovitine or purvalanol A, or genetically, with cyclin B1 siRNA, did not inhibit chemosensitization uniformly across the cell lines. Furthermore, we found that AZD7762 augmented high-intensity γH2AX signaling in gemcitabine-treated cells, suggesting the presence of replication stress when CHK1 is inhibited. Finally, the ability of roscovitine to prevent chemosensitization correlated with its ability to inhibit AZD7762-induced high-intensity γH2AX, but not aberrant pHH3, suggesting that the effects of AZD7762 on DNA replication or repair rather than aberrant mitotic entry determine gemcitabine chemosensitization in pancreatic cancer cells.     

Normal cells, but not cancer cells, survive severe Plk1 depletion

We previously reported the phenotype of depletion of polo-like kinase 1 (Plk1) using RNA interference (RNAi) and showed that p53 is stabilized in Plk1-depleted cancer cells. In this study, we further analyzed the Plk1 depletion-induced phenotype in both cancer cells and primary cells. The vector-based RNAi approach was used to evaluate the role of the p53 pathway in Plk1 depletion-induced apoptosis in cancer cells with different p53 backgrounds. Although DNA damage and cell death can occur independently of p53, p53-deficient cancer cells were much more sensitive to Plk1 depletion than cancer cells with functional p53. Next, the lentivirus-based RNAi approach was used to generate a series of Plk1 hypomorphs. In HeLa cells, two weak hypomorphs showed only slight G2/M arrest, a medium hypomorph arrested with 4N DNA content, followed later by apoptosis, and a strong Plk1 hypomorph underwent serious mitotic catastrophe. In well-synchronized HeLa cells, a medium level of Plk1 depletion caused a 2-h delay of mitotic progression, and a high degree of Plk1 depletion significantly delayed mitotic entry and completely blocked cells at mitosis. In striking contrast, normal hTERT-RPE1 and MCF10A cells were much less sensitive to Plk1 depletion than HeLa cells; no apparent cell proliferation defect or cell cycle arrest was observed after Plk1 depletion in these cells. Therefore, these data further support suggestions that Plk1 may be a feasible cancer therapy target.     

Plk1 phosphorylation of Orc2 promotes DNA replication under conditions of stress

Polo-like kinase 1 (Plk1) plays pivotal roles in mitosis; however, little is known about its function in S phase. In this study, we show that inhibition of Plk1 impairs DNA replication and results in slow S-phase progression in cultured cancer cells. We have identified origin recognition complex 2 (Orc2), a member of the DNA replication machinery, as a Plk1 substrate and have shown that Plk1 phosphorylates Orc2 at Ser188 in vitro and in vivo. Furthermore, Orc2-S188 phosphorylation is enhanced when DNA replication is under challenge induced by ultraviolet, hydroxyurea, gemcitabine, or aphidicolin treatment. Cells expressing the unphosphorylatable mutant (S188A) of Orc2 had defects in DNA synthesis under stress, suggesting that this phosphorylation event is critical to maintain DNA replication under stress. To dissect the mechanism pertinent to this observation, we showed that Orc2-S188 phosphorylation associates with DNA replication origin and that cells expressing Orc2-S188A mutant fail to maintain the functional pre-replicative complex (pre-RC) under DNA replication stress. Furthermore, the intra-S-phase checkpoint is activated in Orc2-S188A-expressing cells to cause delay of S-phase progress. Our study suggests a novel role of Plk1 in facilitating DNA replication under conditions of stress to maintain genomic integrity.     

Polo-like kinase1, a new target for antisense tumor therapy

The Polo-like kinase 1 (Plk1) is a highly conserved mitotic serine/threonine kinase which is commonly overexpressed in cancer cell lines. Plk1 positively regulates mitotic progression by activating the CDC25C-CDK1 amplification loop and by regulating late mitotic events, primarily the ubiquitin-dependent proteolysis. In the present study, an antisense strategy against Plk1 mRNA was developed to specifically inhibit cell proliferation of cancer cells in cell culture and in the nude-mouse tumor model. Among 41 phosphorothioate antisense oligodeoxynucleotides tested, the 20-mer JWG2000 strongly inhibited expression of Plk1 in cultured A549 cells, leading to loss of cell viability, and exhibited anti-tumor activity in nude mice A549 xenograft. JWG2000 did not inhibit growth and viability of primary human mesangial cells and human amnion fibroblasts.     

Polo-like kinases inhibited by wortmannin. Labeling site and downstream effects

Polo-like kinases play crucial roles throughout mitosis. We previously reported that wortmannin potently inhibits Polo-like kinase 1 (Plk1). In this study, we show that wortmannin also strongly inhibits Polo-like kinase 3 (Plk3). To further characterize this inhibition, we identified the sites of labeling on Plk1 and Plk3 targeted by AX7503, a tetramethylrhodamine-wortmannin conjugate. AX7503 labeling on Plk1 and Plk3 was found to occur on a conserved ATP binding site residue. In addition, we show that wortmannin inhibits Plk3 activity in live cells at concentrations commonly used to inhibit the more well known targets of wortmannin, the phosphoinositide 3-kinases. Importantly, we found that inhibition of Plk3 by wortmannin lead to a decrease in phosphorylation of p53 on serine 20 induced by DNA damage, demonstrating the effect of wortmannin on a downstream Plk3 target. Taken together, our results suggest that wortmannin can affect multiple functions of Plk3 in cell cycle progression and at the DNA damage check point. The identification of the labeling sites of Plk1 and Plk3 by AX7503 may be useful in designing more effective compounds to target Polo-like kinases for cancer treatment and also may be useful for the structural study of Plk domains.     

Identification of NME5 as a contributor to innate resistance to gemcitabine in pancreatic cancer cells

The limited therapeutic effect of gemcitabine on pancreatic cancer is largely attributed to pre-existing or acquired resistance of the tumor cells. This study was aimed at screening for candidate resistance-related gene(s) and elucidating the underlying mechanisms. NME5 was found to be highly expressed in an innate gemcitabine-resistant human pancreatic cancer sample and the cell line PAXC002 derived from the sample. Downregulation of NME5 significantly reversed gemcitabine resistance in PAXC002 cells, whereas NME5 overexpression induced gemcitabine resistance in the pancreatic cancer cell line BxPC-3. NME5 attenuated the induction of apoptosis and cell cycle arrest induced by gemcitabine, probably accounting for the blunted sensitivity to gemcitabine. Furthermore, NME5 was demonstrated to play its role in a nuclear factor kappaB (NF-κB)-dependent manner. NME5 was capable of directly binding NF-κB, and possibly regulated its expression level in PAXC002 cells. Our results also suggest that NF-κB is a key executor of NME5 in regulating apoptosis and cell cycle. All of these data suggest that NME5 is a promising target for relieving innate gemcitabine resistance in pancreatic cancer cells.     

DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells

Although gemcitabine is the most effective chemotherapeutic agent against pancreatic cancer, a growing concern is that a substantial number of patients acquire gemcitabine chemoresistance. To elucidate the mechanisms of acquisition of gemcitabine resistance, we developed gemcitabine-resistant cell lines from six human cancer cell lines; three pancreatic, one gastric, one colon, and one bile duct cancer. We first analyzed gemcitabine uptake using three paired parental and gemcitabine resistant pancreatic cancer cell lines (PK-1 and RPK-1, PK-9 and RPK-9, PK-59 and RPK-59) and found that uptake of gemcitabine was rapid. However, no DNA damage was induced in resistant cells. We further examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance and found a remarkable reduction in the expression of deoxycytidine kinase (DCK). DCK is a key enzyme that activates gemcitabine by phosphorylation. Genetic alterations and expression of DCK were studied in these paired parental and derived gemcitabine-resistant cell lines, and inactivating mutations were found only in gemcitabine-resistant cell lines. Furthermore, siRNA-mediated knockdown of DCK in the parental cell lines yielded gemcitabine resistance, and introduction of DCK into gemcitabine-resistant cell lines invariably restored gemcitabine sensitivities. Mutation analyses were expanded to three other different paired cell lines, DLD-1 and RDLD-1 (colon cancer cell line), MKN-28 and RMKN-28 (gastric cancer cell line), and TFK-1 and RTFK -1 (cholangiocarcinoma cell line). We found inactivating mutations in RDLD-1 and RTFK-1 and decreased expression of DCK in RMKN-28. These results indicate that the inactivation of DCK is one of the crucial mechanisms in acquisition of gemcitabine resistance.     

DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells

Pancreatic cancer is one of the most aggressive human malignancies with extremely poor prognosis. The moderate activity of the current standard gemcitabine and gemcitabine-based regimens was due to pre-existing or acquired chemo-resistance of pancreatic cancer cells. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in gemcitabine resistance, and studied the underlying mechanisms. We found that NU-7026 and NU-7441, two DNA-PKcs inhibitors, enhanced gemcitabine-induced cytotoxicity and apoptosis in PANC-1 pancreatic cancer cells. Meanwhile, PANC-1 cells with siRNA-knockdown of DNA-PKcs were more sensitive to gemcitabine than control PANC-1 cells. Through the co-immunoprecipitation (Co-IP) assay, we found that DNA-PKcs formed a complex with SIN1, the latter is an indispensable component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). DNA-PKcs–SIN1 complexation was required for Akt activation in PANC-1 cells, while inhibition of this complex by siRNA knockdown of DNA-PKcs/SIN1, or by DNA-PKcs inhibitors, prevented Akt phosphorylation in PANC-1 cells. Further, SIN1 siRNA-knockdown also facilitated gemcitabine-induced apoptosis in PANC-1 cells. Finally, DNA-PKcs and p-Akt expression was significantly higher in human pancreatic cancer tissues than surrounding normal tissues. Together, these results show that DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells.     

pp32 (ANP32A) expression inhibits pancreatic cancer cell growth and induces gemcitabine resistance by disrupting HuR binding to mRNAs

The expression of protein phosphatase 32 (PP32, ANP32A) is low in poorly differentiated pancreatic cancers and is linked to the levels of HuR (ELAV1), a predictive marker for gemcitabine response. In pancreatic cancer cells, exogenous overexpression of pp32 inhibited cell growth, supporting its long-recognized role as a tumor suppressor in pancreatic cancer. In chemotherapeutic sensitivity screening assays, cells overexpressing pp32 were selectively resistant to the nucleoside analogs gemcitabine and cytarabine (ARA-C), but were sensitized to 5-fluorouracil; conversely, silencing pp32 in pancreatic cancer cells enhanced gemcitabine sensitivity. The cytoplasmic levels of pp32 increased after cancer cells are treated with certain stressors, including gemcitabine. pp32 overexpression reduced the association of HuR with the mRNA encoding the gemcitabine-metabolizing enzyme deoxycytidine kinase (dCK), causing a significant reduction in dCK protein levels. Similarly, ectopic pp32 expression caused a reduction in HuR binding of mRNAs encoding tumor-promoting proteins (e.g., VEGF and HuR), while silencing pp32 dramatically enhanced the binding of these mRNA targets. Low pp32 nuclear expression correlated with high-grade tumors and the presence of lymph node metastasis, as compared to patients' tumors with high nuclear pp32 expression. Although pp32 expression levels did not enhance the predictive power of cytoplasmic HuR status, nuclear pp32 levels and cytoplasmic HuR levels associated significantly in patient samples. Thus, we provide novel evidence that the tumor suppressor function of pp32 can be attributed to its ability to disrupt HuR binding to target mRNAs encoding key proteins for cancer cell survival and drug efficacy.     

Zinc depletion efficiently inhibits pancreatic cancer cell growth by increasing the ratio of antiproliferative/proliferative genes

We investigated the ability of the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to reduce pancreatic cancer cell viability. TPEN was much more efficient to inhibit pancreatic adenocarcinoma cell growth than a panel of anti-cancer drugs, including 5-fluorouracil, irinotecan, cisplatin, edelfosine, trichostatin A, mitomycin C, and gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Moreover, TPEN showed a dose- and time-dependent anti-proliferative effect significantly higher on pancreatic cancer cells than on normal primary fibroblasts. This effect may be explained by a significantly higher zinc depletion by TPEN in pancreatic cancer cells as compared to fibroblasts. Cell viability reduction by TPEN was associated to both G1-phase cell cycle arrest and apoptosis, and to the increased ratio of the expression level of cyclin-Cdk inhibitor versus cyclin genes and apoptotic versus anti-apoptotic genes. Finally, we show that apoptotic cell death induced by TPEN involved mitochondrial injury and caspase 3 and caspase 8 activation. In this study, we suggest that zinc depletion may be an efficient strategy in the treatment of pancreatic cancer because of its reduced antiproliferative effect on normal cells.     

Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints

Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as “authentic” bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents’ gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient’s tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.     

The role of Polo-like kinase 1 in the inhibition of centrosome separation after ionizing radiation

Activation of the G2/M cell cycle checkpoint by DNA damage prevents cells from entering mitosis. Centrosome separation is initiated in G2 phase and completed in M phase. This critical process for cell division is targeted by G2/M checkpoint. Here we show that Plk1 signaling plays an important role in regulation of centrosome separation after DNA damage. Constitutively active Plk1 overrides the inhibition of centrosome separation induced by DNA damage. This inhibition is dependent on ATM, but not on Chk2 or Chk1. Nek2 is a key regulator of centrosome separation and is a target of Plk1 in blocking centrosome separation. We found that Plk1 can phosphorylate Nek2 in vitro and interacts with Nek2 in vivo. Down-regulation of Plk1 with RNA interference prevents Nek2-induced centrosome splitting. DNA damage is known to inhibit Plk1 activity. We propose that the DNA damage-induced inhibition of Plk1 leads to inhibition of Nek2 activity and thus prevents centrosome separation.     

The MAPK-activated protein kinase 2 mediates gemcitabine sensitivity in pancreatic cancer cells

Pancreatic carcinoma is the major clinical entity where the nucleoside analog gemcitabine is used for first-line therapy. Overcoming cellular resistance toward gemcitabine remains a major challenge in this context. This raises the need to identify factors that determine gemcitabine sensitivity in pancreatic carcinoma cells. We previously found the MAPK-activated protein kinase 2 (MK2), part of the p38/MK2 stress response pathway, to be required for DNA replication fork stalling when osteosarcoma-derived cells were treated with gemcitabine. As a consequence, inhibition or depletion of MK2 protects these cells from gemcitabine-induced death (Köpper, et al. Proc Natl Acad Sci USA 2013; 110:16856–61). Here, we addressed whether MK2 also determines the sensitivity of pancreatic cancer cells toward gemcitabine. We found that MK2 inhibition reduced the intensity of the DNA damage response and enhanced survival of the pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and Panc-1, which display a moderate to strong sensitivity to gemcitabine. In contrast, MK2 inhibition only weakly attenuated the DNA damage response intensity and did not enhance long-term survival in the gemcitabine-resistant cell line PaTu 8902. Importantly, in BxPC-3 and MIA PaCa-2 cells, inhibition of MK2 also rescued increased H2AX phosphorylation caused by inhibition of the checkpoint kinase Chk1 in the presence of gemcitabine. These results indicate that MK2 mediates gemcitabine efficacy in pancreatic cancer cells that respond to the drug, suggesting that the p38/MK2 pathway represents a determinant of the efficacy by that gemcitabine counteracts pancreatic cancer.     

A synthetic lethal screen identifies the Vitamin D receptor as a novel gemcitabine sensitizer in pancreatic cancer cells

Overcoming chemoresistance of pancreatic cancer (PCa) cells should significantly extend patient survival. The current treatment modalities rely on a variety of DNA damaging agents including gemcitabine, FOLFIRINOX, and Abraxane that activate cell cycle checkpoints, which allows cells to survive these drug treaments. Indeed, these treatment regimens have only extended patient survival by a few months. The complex microenvironment of PCa tumors has been shown to complicate drug delivery thus decreasing the sensitivity of PCa tumors to chemotherapy. In this study, a genome-wide siRNA library was used to conduct a synthetic lethal screen of Panc1 cells that was treated with gemcitabine. A sublethal dose (50 nM) of the drug was used to model situations of limiting drug availability to PCa tumors in vivo. Twenty-seven validated sensitizer genes were identified from the screen including the Vitamin D receptor (VDR). Gemcitabine sensitivity was shown to be VDR dependent in multiple PCa cell lines in clonogenic survival assays. Sensitization was not achieved through checkpoint override but rather through disrupting DNA repair. VDR knockdown disrupted the cells’ ability to form phospho-γH2AX and Rad51 foci in response to gemcitabine treatment. Disruption of Rad51 foci formation, which compromises homologous recombination, was consistent with increased sensitivity of PCa cells to the PARP inhibitor Rucaparib. Thus inhibition of VDR in PCa cells provides a new way to enhance the efficacy of genotoxic drugs.