Cancer Cell Line LNCaP Research Articles

Abstract A075: Bcl-2 family proteins as drug targets; Can prostate cancer be primed for apoptosis?

Abstract Cancer is a disease of accumulated genetic mutations that alter a cell’s function. One of the common cellular functions affected in cancer cells is the apoptotic pathway. Apoptosis, also known as programmed cell death, is a regulatory biologic function controlled by protein interactions within the BCL-2 protein family. Normally, cells use this pathway to induce cell death when they detect an error in the cell’s functioning that cannot be fixed. In multiple types of cancers, including prostate cancer, there is often an excess of pro-survival BCL-2 proteins. Consequently, these cells have an inability to induce apoptosis, leading to an increase in cancer cell survival, sometimes even in the presence of chemotherapeutic chemicals. This research investigates the potential difference in BCL-2 proteins between metastatic prostate cancer cells and premetastatic prostate cancer cells. Specifically, changes in cancer cell response to chemotherapy after inhibiting antiapoptotic BCL-2 proteins via small-molecule inhibitors are being evaluated. We hypothesize that chemoresistant cancer cells are sensitized to BCL-2 inhibitors by first priming their mitochondria with a traditional chemotherapeutic agent. “Mitochondrial priming” is bringing the antiapoptotic BCL-2 proteins to their apoptotic threshold (1). To accomplish these aims, metastatic prostate cancer cell lines PC3, LnCap C4/2, and DU145 as well as a normal prostate cancer cell line, PNT2, were used. We first sought to compare the level of BCL-2 family proteins among the different cell types. PC3 cells have significantly higher levels of BCL-2, BCL-xL, and BAD as detected by Western blot and quantified by densitometry, corroborating findings from other labs. Next, cells were treated with 5uM etoposide or 100nM docetaxel for 24 hours or 48 hours, and viability was assessed by MTT and apoptosis via Annexin V-FITC. We found that cell viability was significantly decreased in LnCap and DU145 cells at 24 hours, which can be attributed specifically to apoptosis. Moreover, increasing amounts of apoptosis occurred after 48 hours of treatment. Comparatively, PNT2 and PC3 cells have significantly less apoptosis induced by either etoposide or docetaxel treatment. Apoptosis was further confirmed by caspase-3 activation as well as the ability to block apoptosis by pretreatment with the caspase inhibitor Boc-D-fmk in LnCap cells. Additionally, overexpression of BCL-xL in LnCap cells abrogated etoposide-induced apoptosis. Therefore BCL-xL is a potential target for priming. Next, we focused on enhancing the apoptotic effects of etoposide and docetaxel by combing their use with ABT-737, which is a pan BCL-2 antiapoptotic inhibitor. Evaluating the effects of these drugs on various prostate cancerous cell lines in comparison to the effects on a normal prostate cell line may provide insight into their clinical usefulness and relevance in combination treatments. Cells were treated with 0, 100nM, 500nM, 1uM, 2.5uM, or 5uM ABT-737 for 24 hours and apoptosis was assessed via Annexin V-FITC. Preliminary results suggest that on its own, ABT-737 has little effect on LnCap cells (~10%) and no effect on PC3, DU-145, or PNT2 cells. However, pretreating LnCap cells for 24 hours with etoposide as low as 100nM followed by 5 hours exposure to 500nM ABT-737 can induce twice as much apoptosis. These results suggest that etoposide “primes” cells and then the BCL-2 inhibitor pushes them over the edge towards apoptosis. Since less etoposide (100nM) in the combined treatment was effective, as opposed to 500nM when given alone, it may be possible to administer lower amounts of clinically established chemotherapeutics. However, preliminary results also show a ~30% increase in apoptosis in DU145 cells and 50% increase in the normal prostate cell line (PNT2) after combination treatment of 50nM docetaxel for 48 hours followed by 5 hours exposure to 1uM ABT-737. These results suggest that even though lower levels of chemotherapeutics may be effective at killing prostate cancer cells when paired with ABT-737, this combination treatment may be just as harmful to healthy prostate cells. Ongoing studies are also focused on combination treatments with ABT-199, which is selective for antiapoptotic BCL-xL. Reference: 1. Chonghalie TN, et al. Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 2011:334(6059):1129-33. Citation Format: Kaitlyn Walsh, Rachel Wilson, Mary E. McManamy, Victoria Del Gaizo Moore. Bcl-2 family proteins as drug targets; Can prostate cancer be primed for apoptosis? [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A075.

Effect of synthetic small molecule RNA on cell cycle and proliferation of prostate cancer cell

Objective To investigate the effect of dsP21-555 on cell cycle and proliferation of prostate cancer cell line PC-3 and LNCaP. Methods dsP21-555 (experimental group) and dsControl (negative control group) were transfected into PC-3 and LNCaP, respectively. Real-time PCR and Western Blotting were used to detect the expression of p21 mRNA and p21 protein in prostate cancer cells after transfection. The cell cycle distribution was detected by flow cytometry. Cell viability and proliferation were measured by MTT assay and colony forming assay. Results The level of p21 mRNA in PC-3 and LNCaP cells was increased to 2.90-fold (P<0.01) and 2.05 - fold (P<0.01) respectively after transfection with dsP21-555. Western Blotting results were consistent with this trend. Flow cytometry showed that the percentage of cells in S phase and G2 / M phase decreased after transfection with dsP21-555, and the proportion of cells in G0 / G1 phase increased. MTT assay showed that the viability of PC-3 and LNCaP cells significantly decreased after dsP21-555 transfection compared with dsControl group. The colony formation assay showed that the number of colonies in the dsP21-555 group was fewer and the cell proliferation ability decreased. Conclusions Synthesized dsP21-555 can significantly activate the expression of p21 gene in prostate cancer cells and significantly inhibit the progression of cell cycle and proliferation ability of prostate cancer cells. Key words: Prostatic Neoplasms; RNA; Cell Cycle; Cell Proliferation

Immobilization platform to induce quiescence in dormancy-capable cancer cells

Cancer dormancy emerges when tumor cells cease to proliferate but remain alive in a quiescent state. Recent evidence suggests that cancer cells can stay dormant in a patient’s body for years before returning to a proliferative state, leading to cancer relapse. The lack of a system to efficiently identify and study dormant cancer cells is currently limiting further diagnostic and treatment developments to prevent cancer relapse. Herein, we present a novel encapsulation platform to identify and study dormancy-capable cancer cells in a quiescent state by inhibiting proliferation through physical confinement. The platform involves the encapsulation of cells within a stiff silica-PEG hydrogel produced by a sol–gel technique. Cells are immobilized in a nondegradable microenvironment where proliferation and movement are inhibited due to physical confinement of the gel. The platform was tested using non-cancerous cell lines HFF, HUVEC, Jurkat, MEF, and MCF-10A, and cancer cell lines LnCAP, MCF-7, MCF10DCIS.com , MDA-MB-468, and OVCAR-5. Viability and metabolic activity measurements showed that MCF-7, LnCAP, and MCF10DCIS.COM cells remained metabolically active for up to 3 weeks while non-cancerous lines and the rest of the cancer cell lines did not survive after a few days. Ki-67 immunofluorescent staining confirmed that surviving MCF-7 cells underwent cell cycle arrest as early as 48 hours after encapsulation. Furthermore, following extraction and recovery, these cells resumed proliferation, indicating that the induced cell cycle arrest was reversible. These results conclude that physically inhibiting proliferation via the silica-PEG hydrogel system can be used to identify cells that can enter a quiescent state, setting the groundwork for this platform to be explored as a cancer cell dormancy model.

Abstract 3779: Incorporating in-line sample pre-enrichment with FACS and single cell mRNA-seq to facilitate isolation and characterization of prostate cancer circulating tumor cells

Abstract For decades, circulating tumor cells (CTCs) have been used as biomarkers to indicate disease progression and survival in patients with metastatic tumors. Detection of CTCs is an attractive, non-invasive blood test for early diagnosis of many types of cancer. However, CTCs are present at extremely low frequency in blood, and it is challenging to recover them in typical multi-step sample processing workflows. EpCAM, the widely accepted marker for CTC, has been found being down regulated in protein expression and RNA expression analysis in many cases thus EpCAM-dependent CTC detection might not be sensitive enough. We have developed a new sample pre-enrichment technology incorporated in line with fluorescence activated cell sorting (BD FACS™) platforms to demonstrate a one-step sample processing workflow to minimize cell loss and maximize CTC recovery. The pre-enrichment technology combines magnetic depletion of white blood cells and acoustic removal of lysed red blood cells, sending enriched tumor cells directly to a cell sorter for single CTC isolation. The in-line enrichment and sorting technology has shown 90% removal of CD45+ cells and over 90% recovery of target cells going through the system. Furthermore, we have incorporated molecular indexing technology for next generation sequencing (NGS) into the single CTC isolation to streamline gene expression analysis. We conducted a pilot study in which prostate cancer cell lines PC3, DU145, and LNCaP were spiked in healthy donors’ peripheral blood, quantitatively recovered, and individually sorted into 96-well BD™ Precise barcoded plates using a BD Influx™ sorter equipped with the in-line pre-enrichment module. The sorted cells were then sequenced for a prostate cancer targeted gene panels with approximate 100 genes, including EpCAM. Single cell sorting and sequencing were also performed on cell line cells directly and confirmed the workflow did not affect the gene expression. In LNCaP and PC3 cells, EpCAM was not the high expression marker in the genes analyzed, and its protein expression was also heterogeneous. The highly expressed genes discovered by single cell mRNA sequencing such as GSTP1 and CAV1 were investigated to provide additional cell surface markers for more sensitive CTC detection. Finally, CTCs from blood samples collected from prostate cancer patients were isolated based on marker panels beyond EpCAM and characterized at the single cell level. The differences between gene expression profiles of patient sample CTCs and cancer cell line cells were revealed. Citation Format: Liping Yu, Silin Sa, Michael Tycon, Xiaoyang Alice Wang. Incorporating in-line sample pre-enrichment with FACS and single cell mRNA-seq to facilitate isolation and characterization of prostate cancer circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3779. doi:10.1158/1538-7445.AM2017-3779

Abstract 2787: Expression of Olfactomedin 4 is regulated by methylation in the benign prostate epithelial cell-RWPE1 and PC-3 prostate cancer cells

Abstract Expression of Olfactomedin 4 gene (OLFM4) plays a critical tumor suppressor role in human and murine prostate cancer. We have previously reported that loss or reduced OLFM4 expression is associated with the progression of human prostate cancer. We and others have further determined that deletions within the OLFM4 gene are present in about 10 to 25% of human prostate cancer samples. However, we have observed that loss of expression of OLFM4 were found in more than 58% of the samples obtained from advanced prostate cancer patients. Therefore, we sought to study the role of epigenetic factors in the regulation of OLFM4 gene expression in normal and prostate cancer cell lines. Methylation of CpG sites in the promoter region of key genes is an important epigenetic mechanism in cancer development. We examined the methylation status of CpG sites in the OLFM4 promoter region in the benign immortalized prostate epithelial cell line (RWPE1), androgen-responsive cancer cells (LNCaP, VCaP), and androgen-independent cancer cells (PC-3 and DU145). There are eight CpG sites (-681, -666, -562, -486, -91, +4, +34) in the promoter regions of OLFM4 gene. The pattern of methylation status was detected and compared between RWPE1 cell and cancer cell lines LNCaP, VCaP, PC-3 and Du145. To test whether hypermethylation of CpG sites of OLFM4 gene can block the expression of OLFM4 gene, RWPE1 cells were treated with 5-azadeoxycytidine (5-Aza.) (5 μM) from 24h to 96h and PC-3 cells were treated with 5-azadeoxycytidine (5-Aza.) (5 μM) for 3 days and 7 days. The methylation status of CpG sites was compared in the presence or absence of 5-Aza. at different time points. The addition of 5-Aza. consistently reduced the percent methylation of the CpG sites. Correspondingly, the expression of the OLFM4 gene was induced after treatment with 5-Aza. in both RWPE1 and PC-3 cells. These results suggest that expression of OLFM4 is regulated by the methylation status of OLFM4 gene in the RWPE1 and PC-3 prostatic cell lines. Citation Format: Hongzhen Li. Expression of Olfactomedin 4 is regulated by methylation in the benign prostate epithelial cell-RWPE1 and PC-3 prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2787.

Abstract 2624: Possibility of prostate cancer prevention based on cancer stem cell theory

Abstract Cancer stem cells are considered to be one of the causes of cancer. In recent years, stemness (stem like) of gene expression patterns in the development process of cancer has been observed. Stemness and pathology of cancer are closely related. Elucidation of the stemness regulation of cancer is an important key to the development of new cancer prevention methods. However, as far as we know, there exist no cancer prevention studies focusing on cancer stem cells. In this study, we have investigated the possibility of prostate cancer prevention with a focus on cancer stem cells. We utilized prostate cancer cell lines PC3, DU145 (androgen-independent) and LNCaP (androgen-dependent) cells. Then, using spheroid formed cells, mRNA expression levels of cancer stem cells markers were estimated by RT-real time-PCR. Cancer cells have several malignant phenotypes under hypoxia, and we investigated for the cancer stem cells induced malignant alteration under hypoxia. In addition, under hypoxia, we investigated cell viability effect under tocotrienol-rich fraction (TRF) from annatto. TRF almost consists of delta- tocotrienol (δ-T3) that has the strongest anti-cancer activity. We observed spheroid formation capacity and compared the spheroid sizes and mRNA levels of DU145, PC3 and LNCaP cells. Size of DU145 spheroid is smaller in comparison to spheroids of PC3 and LNCaP. mRNA expression levels of cancer stem cells marker were elevated in PC3 and LNCaP cells. This means that PC3 and LNCaP cells have higher concentration of cancer stem cells. Furthermore, stemness decreases with increase of passage number, the number of cultures. We confirmed the presence of stemness in the formation capacity of spheroid until passage 4. Under hypoxia, cell viability of cancer stem cells was elevated compared to normal cultured prostate cancer cells. In addition, under hypoxia and TRF treatment, cell viability of cancer stem cells decreased with increase in TRF concentration compared to control (0 μg/ml). Our results suggest that TRF suppress cell viability of prostate cancer stem cells under hypoxic states and is effective in prostate cancer prevention. Citation Format: Nobuya Shiozawa, Ryosuke Sugahara, Ayami Sato, Masako Ota, Tomohiro Yano. Possibility of prostate cancer prevention based on cancer stem cell theory. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2624.

Abstract 4964: The isolation of prostate cancer specific circulating tumor cells in the blood of patients with metastatic castration-resistant prostate cancer

Abstract Background: Circulating tumor cells (CTC) consist of a heterogeneous population of very rare cells of the primary tumor or its own metastases. A growing amount of evidence has shown that subpopulations of carcinoma cells undergo epithelial-to-mesenchymal transitions (EMT), resulting in their increased motility which facilitates their intravasation into the blood circulation. Gold standard of CTC-isolation is the CellSearchSystem based on epithelial cell adhesion molecule (EpCAM) enrichment, though it is known that EpCAM is downregulated in al advanced prostate cancers and in all CTCs. Furthermore these CTC isolation techniques are not able to isolate organ specific CTCs. The aim of this pilot study is the development of a prostate cancer specific functionalized wire, which detects prostate cancer specific circulating tumor cells (PCTC) in metastatic PCa patients. The CTC detection rate will be compared between an EpCAM functionalized wire and a PCa specific functionalized wire. Methods: We combined 4 antibodies against Prostate specific membrane antigen (PSMA), Prostate specific antigen (PSA), Prostate specific stem cell antigen (PSCA) and epithelial cell adhesion molecule (EpCAM). Evaluations of these antibodies were performed by immunofluorescence analysis of the human prostate cancer cell lines PC3 and LNCaP. PCa specific functionalization of the wire was determined with spiking experiments. Blood samples from healthy donors were spiked with PC-3 and LNCaP cells to test the wire for cell-binding. Afterwards blood of 15 metastatic castration-resistant prostate cancer (CRPC) patients with metastatic progression, documented by prostate-specific antigen or radiologic criteria, was investigated. These samples were simultaneously analyzed in the flow system with two different functionalized wires. The captured CTC and PCTC were identified by immunofluorescence staining using pan cytokeratin and Hoechst-33258 positive signals as well as CD45 negative criteria. Results: The spiking experiments indicate that a sensitive ex vivo isolation with different functionalized wires is possible. In summary, PCTC counts ranged from 0-122 per PCTC (median 9) and CTC counts ranged from 0-22 per CTC (median 3). Our data shows that a more sensitive isolation of PCTC′s is possible using prostate cancer specific functionalized wire compared to EpCAM functionalized wire (p ≤ 0,001). The CTC isolation sensitivity was about 86% for PCa-functionalized wire and about 73% for EpCAM functionalized wire. Conclusions: PCTC can be isolated with prostate cancer-specific functionalization of the wire. Simultaneously CTC that underwent EMT can be isolated. These organ specific subpopulations might have a prognostic effect on the clinical outcome and on the development of personalized medicine. This proof of concept shows how important it is to optimize the EpCam based CTC-detection. Citation Format: Gerit Theil, Stefanie Schmidt, Kersten Fischer, Klaus Lücke, Paolo Fornara. The isolation of prostate cancer specific circulating tumor cells in the blood of patients with metastatic castration-resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4964.

Abstract 555: Prostate cancer cell-induced differentiation of human monocytes into MDSCs ex vivo is inhibited by targeting STAT3

Abstract Background: The transcription factor Signal Transducer and Activator of Transcription Factor 3 (STAT3) is implicated in acquired drug resistance, metastases and immune suppression in prostate cancer and is a potential target in drug-resistant prostate cancer. Today, the treatment options for metastatic drug-resistant prostate cancer are very limited and existing immunotherapies have so far shown low efficacy. Cancer cells may avoid immune responses by expressing immunosuppressive markers or activating immunosuppressive cells. Myeloid-derived suppressor cells (MDSC) play a major role in the suppression of antitumor immunity and active STAT3 signaling has been shown to be involved in the immunosuppressive functions of these cells. Elevated levels of MDSC have been found in the peripheral blood and at the tumor site of prostate cancer patients and to correlate with disease progression. Aims: In this study we aimed to investigate if human monocytes could be induced to differentiate into immunosuppressive cells in the presence of prostate cancer cells and if this effect could be blocked by the STAT3 inhibitor GPA500 (galiellalactone). Methods: Monocytes were isolated from peripheral blood mononuclear cells from healthy human donors using a magnetic monocyte enrichment kit. Monocytes were co-cultured with the prostate cancer cell lines DU145, LNCaP or longterm IL6 treated LNCaP cells (LNCaP-IL6+) in transwells allowing cancer cells and monocytes to share culture medium. The co-cultures were grown for three days with or without the STAT3 inhibitor GPA500 (5 uM or 10 uM) with subsequent analysis by flow cytometry for cell surface markers of MDSC differentiation (CD14+ HLA-DRlo). The presence of active STAT3 (pSTAT3) in the prostate cancer cell lines was determined by Western blot. Results: Monocytes in the presence of DU145 and LNCaP-IL6+ cells showed a significantly increased population of CD14+ HLA-DRlo cells. In the presence of GPA500 the induction of this population was inhibited in a dose dependent manner compared to untreated cells. Co-culture with LNCaP cells that lack pSTAT3 did not induce this population in monocytes after three days in culture. Furthermore, GPA500 induced a dendritic cell-like population in monocytes in the absence of prostate cancer cell co-culture. This population was also observed in monocytes when co-cultured with LNCaP-IL6+ cells, but not in DU145 co-cultures, with GPA500 present. Conclusion: The prostate cancer cell lines DU145 and LNCaP-IL6+ were able to induce a monocyte population displaying MDSC markers and this induction could be inhibited by the STAT3 inhibitor GPA500. Impact: This study demonstrates that inhibiting the transcription factor STAT3 in prostate cancer could potentially reverse the immunosuppressive mechanisms caused by MDSC activation and that STAT3 inhibitors, alone or in combination with other therapies, could be a new treatment option for prostate cancer. Citation Format: Rebecka Hellsten, Karin Leandersson, Anders Bjartell, Martin E. Johansson. Prostate cancer cell-induced differentiation of human monocytes into MDSCs ex vivo is inhibited by targeting STAT3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 555.

Abstract 5171: Interleukin-17 acts through MMP7 to promote prostate cancer

Abstract Interleukin-17 (IL-17) is a key cytokine in inflammation, which has been shown to play critical roles in development of prostate cancer, skin cancer, colon cancer, and breast cancer. We have previously demonstrated that IL-17 promotes prostate carcinogenesis, accompanied with increased expression of matrix metalloproteinase 7 (MMP7) in the mouse prostate. The purpose of the present study was to determine whether MMP7 mediates IL-17's action and the underlying mechanisms. Mmp7 knockout (Mmp7−/−) mice were crossbred with phosphatase and tensin homolog (Pten) conditional knockout mice (Ptenpc-/−, prostate-specific through probasin promoter-driven Cre recombinase). The prostate tumor phenotypes were analyzed among Mmp7+/+;Ptenpc-/−, Mmp7+/−;Ptenpc-/−, and Mmp7−/−;Ptenpc-/− mice. MMP7-overexpression or MMP7 knockdown using small interfering RNA (siRNA) was generated in human prostate cancer LNCaP and C4-2B cell lines to explore the molecular mechanisms of MMP7's actions. We found that Mmp7−/−;Ptenpc-/− mice displayed prostates that were smaller than Mmp7+/+;Ptenpc-/− or Mmp7+/−;Ptenpc-/− mice. Mmp7−/−;Ptenpc-/− mice developed a reduced number of invasive prostate adenocarcinomas with lower rate of cellular proliferation and higher rate of apoptosis than Mmp7+/+;Ptenpc-/− or Mmp7+/−;Ptenpc-/− mice. In addition, the invasiveness of prostate tumor cells isolated from Mmp7−/−;Ptenpc-/− mice was significantly reduced compared to those isolated from Mmp7+/+;Ptenpc-/− mice in Matrigel® invasion assays. Expression of the epithelial-to-mesenchymal transition (EMT) markers, such as β-catenin, vimentin, Snail, and Slug, was dramatically reduced in Mmp7−/−;Ptenpc-/− mice compared to Mmp7+/+;Ptenpc-/− or Mmp7+/−;Ptenpc-/− mice. In contrast, expression of the epithelial markers such as E-cadherin and claudin was increased in Mmp7−/−;Ptenpc-/− mice compared to Mmp7+/+;Ptenpc-/− or Mmp7+/−;Ptenpc-/− mice. MMP7 cleaved E-cadherin and disrupted E-cadherin/β-catenin complex to increase cytoplasmic and nuclear β-catenin levels in the prostate cancer cells, thus up-regulating EMT transcription factors. In addition, IL-17 receptor C (Il-17rc) and Pten double knockout (Il-17rc−/−;Ptenpc-/−) mice echoed the reduction of EMT in Mmp7−/−;Ptenpc-/− mice, compared to Il-17rc+/+;Ptenpc-/− mice. Further, IL-17 treatment induced MMP7 expression in LNCaP and C4-2B cells, leading to increased expression of EMT markers. In contrast, MMP7 knockdown inhibited IL-17-induced expression of EMT markers. Taken together, our results demonstrate that MMP7 mediates IL-17's function in promoting prostate carcinogenesis through induction of EMT. This work was partially funded by National Institutes of Health (R01CA174714 and P20GM103518), Department of Defense (W81XWH-14-1-0050, W81XWH-14-1-0149, W81XWH-14-1-0458, and W81XWH-15-1-0444), and Tulane's Institute of Integrated Engineering for Health and Medicine. Citation Format: Qiuyang Zhang, Sen Liu, Keshab Parajuli, Zongbing You. Interleukin-17 acts through MMP7 to promote prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5171.

Abstract A92: Aminomethylphosphonic acid inhibits human prostate xenograft tumor growth through interfering glycine synthesis in the cancer cells

Abstract Rapidly proliferating cancer cells consume more glycine than rapidly proliferating normal cells, which offers an opportunity to target glycine metabolism in the cancer cells while avoiding causing severe side effects in the normal cells. Two-thirds of glycine consumed is synthesized intracellularly, e.g., through conversion of serine into glycine by serine hydroxymethyltransferase (SHMT). Aminomethylphosphonic acid (AMPA, C3H3NO5P) is an analog to glycine, which inhibits SHMT enzyme activity, thus blocking conversion of serine into glycine. Our previous in-vitro studies have shown that AMPA inhibited cell growth in six cancer cell lines (including four human prostate cancer cell lines PC-3, DU-145, LNCaP, and C4-2B), while had little effects on two human normal immortalized prostatic epithelial cell lines (RWPE-1 and pRNS-1-1). The purpose of the present study was to determine if AMPA could inhibit PC-3 xenograft tumor growth in nude mice. PC-3-LacZ-luc cells (stably expressing LacZ and luciferase for staining and in-vivo imaging) were first implanted subcutaneously to form tumors. Then, the tumors were cut into 2-mm pieces and surgically implanted orthotopically in the prostates of 39 six-week-old athymic nude male mice that were castrated during the same surgery. One week later, the prostate tumor sizes were measured using D-luciferin and IVIS® Lumina XRMS imaging system (PerkinElmer, Inc.). The animals were randomized into three treatment groups: 1) saline as control (n = 14); 2) 400 mg/kg/day of AMPA (n= 10); and 3) 800 mg/kg/day of AMPA (n = 15). The treatment was administrated intraperitoneally once a day until animal death. Animal body weight measurement and in-vivo imaging were performed once every 5 to 7 days. Upon animal death, tumors were harvested and weighed. Pathologic examination, immunohistochemical staining, and Western blot analyses were performed using the tumors. We found that the tumor size rapidly increased in the control group, whereas the tumor size increased only slightly in the two AMPA-treated groups (p &amp;lt; 0.05). Animal survival time was significantly longer in the AMPA-treated groups than the control group (p &amp;lt; 0.05) and the high-dose group had slightly longer survival time than the low-dose group (p = 0.087). The average final tumor weight was significantly less in the high-dose group than the control group (p &amp;lt; 0.05). The levels of cellular inhibitor of apoptosis protein 1 (C-IAP1) and cyclin D1 were dramatically reduced in the tumors from the two AMPA-treated groups compared to the control group. We also observed that the two AMPA-treated groups had much less intra-abdominal metastases compared to the control group. Further in-vitro studies found that AMPA inhibited migration and invasion of PC-3 cells using Transwell assays. In conclusion, we found that AMPA inhibits human prostate xenograft tumor growth through inducing apoptosis and inhibiting cellular proliferation, which suggests that AMPA may be developed into a new treatment for prostate cancer. Citation Format: Keshab R. Parajuli, Qiuyang Zhang, Sen Liu, Zongbing You. Aminomethylphosphonic acid inhibits human prostate xenograft tumor growth through interfering glycine synthesis in the cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A92.

Abstract 4649: Apigenin increases maspin expression and suppresses invasiveness in prostate cancer cells

Abstract Acquisition of metastatic capability during prostate cancer progression leads to clinically incurable malignancy and accounts for the majority of deaths from this disease. Metastasis involves multiple processes, including invasion, intravasation, extravasation and tumor growth at the secondary site. Maspin, a unique member of the serpin (serine protease inhibitor) family, shown to regulate cell motility, invasion and contributes in tumor metastasis. Loss of maspin has been frequently identified in clinical prostate cancer specimens and prostate cancer cell lines, therefore novel therapeutic approaches to restore its expression are needed. Apigenin (4′, 5, 7-trihydroxyflavone), a plant flavone has shown to possess anticancer properties and alters pathways that regulate tumor cell invasion and metastasis, however molecular basis of these effects remains unclear. We demonstrate that apigenin enhances maspin expression in human prostate cancer cells through upregulation of p53 activity and downregulation of histone deacetylase (HDAC) activity. Treatment of human prostate cancer LNCaP cells (harboring wild type p53) with 1-40 μM apigenin for 72 h resulted in dose- and time- dependent decrease in cell invasion and migration with concurrent increase in maspin expression and increased its nuclear localization. Apigenin treatment also resulted in the inhibition of HDAC enzyme activity, increase in H3 and H4 acetylation and p53 activation, similar with the use of Class I HDAC inhibitor, trichostatin A. In human prostate cancer DU145 cells, harboring mutant p53, apigenin treatment resulted in an increase in maspin expression which correlated with upregulation of wild-type p53 activity, conversely trichostatin A was unable to upregulate maspin expression. Transfection of DU145 cells with maspin reporter plasmid increased the activity of maspin promoter, consistent with the results. In another approach, endogenous levels of mapsin was restored after transfection of cells with wild type p53 in prostate cancer PC-3 cells (p53 null), which was further increased by apigenin treatment. Furthermore, knockdown of HDAC1 did not affect maspin levels in human prostate cancer LNCaP and DU145 cell lines suggesting that HDAC1 inhibition does not play a role in maspin regulation, whereas it appears that apigenin-dependent global modulation of HDAC activity resulted in decreased cell invasion and proliferation. Taken together, our findings suggest that apigenin-mediated increase in maspin expression is regulated, in part, by p53 and its activation resulting in decreased invasiveness and migration capabilities of prostate cancer cells. Citation Format: Sanjeev Shukla, Ata Abbas, Sanjay Gupta. Apigenin increases maspin expression and suppresses invasiveness in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4649. doi:10.1158/1538-7445.AM2015-4649

Abstract 2654: Human antibodies specific for oxidized macrophage migration inhibitory factor (oxMIF) synergize with chemotherapeutic agents in animal models of cancer

Abstract Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with pleiotropic biologic effects and is pathologically associated with several types of cancer. oxMIF is an immunological and conformational distinct isoform of MIF that can be mimicked in vitro by mild oxidation of recombinant MIF. Fully human monoclonal antibodies that specifically target oxMIF were shown to inhibit downstream signaling pathways associated with tumor proliferation and progression in vitro and in vivo. We assessed the potential of anti-oxMIF antibodies combined with standard chemotherapeutic agents on the proliferation of cancer cell lines in vitro and in vivo in xenograft models. In vitro, anti-oxMIF sensitization of the prostate cancer cell lines PC3 or LnCAP significantly reduced the half maximal effective concentration (EC50) of mitoxantrone by 30% and 40%, respectively. In the ovarian cancer cell lines A2780, EC50 of cisplatin was reduced by 70% after sensitization with anti-oxMIF. In vivo, mice bearing pancreatic cancer xenografts (COLO357, BxPC3 or MiaPaCa2) treated with anti-oxMIF in combination with gemcitabine had a strong survival advantage compared with mice treated with either monotherapy. This effect was accompanied by a decrease of tumor volumes up to 70% and an increase in the expression of caspase 3 in mice bearing COLO357 xenografts. In addition, combining gemcitabine with anti-oxMIF treatment normalized blood vessel density inside the tumors, decreased their permeability by 25%, and decreased tumor VEGF levels by 40%, whereas gemcitabine alone had the opposite effect. In mice bearing the ovarian human cancer cell line A2780 xenografts, combining anti-oxMIF antibodies with cisplatin led to a 45% reduction of tumor weights. Anti-oxMIF specific antibodies decreased proliferation, angiogenesis and inflammation inside tumor xenografts, but were not cytotoxic (no antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity). These experiments demonstrated synergistic effects between anti-oxMIF antibodies and cytotoxic chemotherapy. Combination treatments were superior to the corresponding monotherapies and led to a stronger inhibition of tumor growth and angiogenesis. A phase 1 clinical study of a novel human antibody that selectively targets oxMIF is currently ongoing in patients with solid malignancies (ClinicalTrials.gov identifier: NCT01765790). Citation Format: Patrice Douillard, Thorsten Hagemann, Michael Freissmuth, Michael Thiele, Alexander Schinagl, Dirk Völkel, Friedrich Scheiflinger, Randolf Kerschbaumer. Human antibodies specific for oxidized macrophage migration inhibitory factor (oxMIF) synergize with chemotherapeutic agents in animal models of cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2654. doi:10.1158/1538-7445.AM2014-2654

Abstract 2397: Nanoformulations of PARP inhibtors for cancer therapy

Abstract Introduction: Poly ADP Ribose Polymerase inhibitor therapy (PARPi) exploits a synthetic lethality strategy in cancers specifically endowed with inherent damage in DNA repair or transcription pathways. Olaparib and BMN-673 are potent PARP inhibitors that are currently indicated for oral PARPi in several clinical trials for a variety of cancers. Oral administration in general results in poor bioavailability and tumor accumulation. Here we report novel and well characterized nanoformulations customized for olaparib (NanoOlaparib) and BMN-673 (NanoBMN) , thus enabling a platform which provides a safe vehicle for intravenous delivery specifically targeted to the tumor, thereby increasing the bioavailability while reducing systemic toxicity. Our nanoplatform is also tailored for the combinatorial chemotherapy and radio-sensitization in several cancers including prostate, ovarian and breast cancer cell lines with and without the BRCA mutations. Methods: Two nanoparticle formulations NanoOlaparib and NanoBMN have been successfully formulated and tested in vitro on several cancer cell lines. The initial combination studies were performed with Cisplatin and PARPi nanoformulations defined by ∼163 nm diameter, zeta potential ∼ +10mV, and loaded with olaparib (2.3mM) or BMN-673 (200µM) and cisplatin (∼3.3mM). Dose response curves over a dynamic range of nanoPARPi therapy on several cell lines were generated using MTS assay and EC50's were determined using Prism. The synergism due to radiosensitization using 220 keV beam was studied for both therapies using isobolograms developed from clonogenic assays on prostate and breast cancer cells. The efficacy of combination PARPi and Pt therapy with nanoparticle platform was determined on ovarian cancer cell lines including PA-1, KURAMOCHI etc. The functionality of nanoPARPi/combination formulations on all the cancers, reflected by inhibition of PARylation, γ-H2AX and RAD-51 was determined by immunoflourescence assays. Results: Radiosensitization showed strong radiosensitization, achieving long-term cell kill of more than 90% with NanOlaparib and 4 Gy radiation in prostate cancer PC3, VCaP and LNCaP cell lines. VCaP which carries a TMPRSS2: ERG fusion was more responsive than PC3 for monotherapy using NanoOlaparib alone, and had better radiation sensitization compared to the other cell lines tested so far. In all studies NanoOlaparib showed better efficacy in combination with radiation than free Olaparib. Similar studies with nanoBMN are underway. Conclusions: Robust nanoparticle formulations NanoOlaparib and NanoOlaparibPt have been successfully demonstrated. We observed a significant enhancement in the efficacy with both nanoformulations. Strong radiosensitization was observed after several days. These results imply an important role for the nanoOlaparib and nanoBMN formulations as chemo and radio-sensitizers enabling several therapeutic approaches. Citation Format: Shifalika Tangutoori, Paige Baldwin, Houari Korideck, Robert Cormack, Mike G. Makrigiorgos, Srinivas Sridhar. Nanoformulations of PARP inhibtors for cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2397. doi:10.1158/1538-7445.AM2014-2397

Stepwise Combination of Multi-Pathway Blockade in Addition to Radio-Chemotherapy for Glioblastoma

Materials/Methods: A PSMA-targeted Glu-Lys urea moiety was conjugated to variably modified DNAPK21, 5’NH2-modified DNAPK27 and 3’NH2-modified DNAPK27 siRNA by amide bond formation from the corresponding active ester. Conjugations used at least a 5:1 ratio of GluLys urea to siRNA. Lipofectamine transfection of the conjugates into LNCaP cells was performed to investigate cellular processing of the siRNA. Treatment without lipofectamine was used to determine if conjugates could independently bind PSMA, internalize and reduce DNAPK mRNA. mRNA levels were determined by RT-PCR. Results: Preliminary results of lipofectamine transfected urea-siRNA conjugates reveal up to 90% knockdown of DNAPK mRNA expression relative to control. Direct treatment of the LNCaP cancer cell line with the urea-5’DNAPK27 conjugate resulted in a 53% DNAPK mRNA knockdown (P Z 0.068), while the urea3’DNAPK27 conjugate demonstrated a 37.6% DNAPK mRNA knockdown (P Z 0.025). The urea-DNAPK21 conjugate yielded 43% DNAPK mRNA knockdown (P<0.01). Conclusions: Cancer cell specific radiosensitization promises to enable tumor control while reducing radiation-induced side effects. We provide results supporting that PSMA-targeted DNAPK siRNA conjugates can be constructed, delivered and successfully targeted to prostate cancer, resulting in reductions in target mRNA in a fashion that may lead to selective prostate cancer radiosensitization. Author Disclosure: M.H. Khattab: None. R.R. Raval: None. Y. Zhang: None. S. Regter: None. M. Hedayati: None. S.R. Banerjee: None. H. Zhou: None. D.V. Ferraris: None. J. Alt: None. C. Rojas: None. B.S. Slusher: None. M.G. Pomper: None. T.L. DeWeese: None.

Synthesis of a new bis(indolyl)methane that inhibits growth and induces apoptosis in human prostate cancer cells

The synthesis and the antiproliferative activity against the human breast MCF-7, SkBr3 and the prostate LNCaP cancer cell lines of a series of bis(indolyl)methane derivatives are reported. The synthesis of new compounds was first accomplished by the reaction of different indoles with trimethoxyacetophenone in the presence of catalytic amounts of hydrochloric acid. A second procedure involving the use of oxalic acid dihydrate [(CO2H)2·2H2O] and N-cetyl-N,N,N-trimethylammonium bromide in water was carried out and led to better yields. Compound 5b significantly reduced LNCaP prostate cancer cell viability in a dose-dependent manner, with an IC50 of 0.64 ± 0.09 μM. To determine whether the growth inhibition was associated with the induction of apoptosis, treated cells were stained using DAPI. LNCaP cells treated with 1 μM of 5b showed the morphological changes characteristic of apoptosis after 24 h of incubation.

Liquid biopsy-based assays to monitor residual disease in cancer.

11096 Background: One of the main challenges in cancer management is monitoring the non-organ-confined disease post metastasis. Recently analysis of circulating tumor nucleic acids has generated a new paradigm for monitoring the progression and molecular pathology of the residual disease through a non-invasive test. Methods: We have developed a new sequencing workflow, which increases the sensitivity and specificity of detecting and quantifying rare tumor-derived nucleic acids among healthy fragments by at least ten-fold. Unlike conventional sequencing library preparation protocols, the majority of extracted circulating DNA fragments are hybridized to sequencing flow cells with minimal modifications. The sequencing data are processed using our proprietary bioinformatics pipelines to search for rare genetic abnormalities within the heterogeneous circulating fragments. Results: To study the performance of our technology, we first evaluated its sensitivity in analytical samples. We spiked varying amounts of LNCaP cancer cell line gDNA into a background of normal gDNA and were able to successfully detect somatic mutations down to 0.1% sensitivity. Subsequently, we investigated the correlation of circulating DNA and tumor gDNA in human xenograft models in mice. In seven different mice models of two different human tumors, we found very strong correlation between somatic mutations detected in all pairs of tumor gDNA and mouse blood cfDNA. After preclinical studies, we initiated a pilot study on human samples across different cancer types. We found more than 90% correlation between tumor mutations in stage IIIb CRC cancer patients and the mutations detected in their circulation (n=7). In a pilot study in prostate cancer patients (n=3), we detected chromosomal abnormalities in multi locations in circulating DNA genome in both advanced and post-relapse stages of the disease. Conclusions: The present work indicates the potential of using circulating nucleic acids to facilitate the integration of real-time molecular pathology into routine cancer care. Our assay will be used to identify residual disease after neoadjuvant chemotherapy and/or surgery, but may also identify a subset of patients who may benefit from alternative therapies.

Fate of the human Y chromosome linked genes and loci in prostate cancer cell lines DU145 and LNCaP

BackgroundProstate cancer is a known cause of mortality in men worldwide although the risk factor varies among different ethnic groups. Loss of the Y chromosome is a common chromosomal abnormality observed in the human prostate cancer.ResultsWe screened 51 standard sequence tagged sites (STSs) corresponding to a male-specific region of the Y chromosome (MSY), sequenced the coding region of the SRY gene and assessed the status of the DYZ1 arrays in the human prostate cancer cell lines DU145 and LNCaP. The MSY was found to be intact and coding region of SRY showed no sequence variation in both the cell lines. However, DYZ1 arrays showed sequence and copy number variations. DU145 and LNCaP cells were found to carry 742 and 1945 copies of the DYZ1, respectively per 3.3 pg of genomic DNA. The DYZ1 copies detected in these cell lines are much below the average of that reported in normal human males. Similarly, the number of “TTCCA” repeat and its derivatives within the DYZ1 arrays showed variation compared to those of the normal males.ConclusionsClearly, the DYZ1 is maximally affected in both the cell lines. Work on additional cell lines and biopsied samples would augment our understanding about the susceptibility of this region. Based on the present work, we construe that copy number status of the DYZ1 may be exploited as a supplementary prognostic tool to monitor the occurrence of prostate cancer using biopsied samples.

Abstract 2972: Pharmacological reactivation of epigenetically regulated genes in prostate cancer.

Abstract Transcriptional silencing caused by promoter hypermethylation is a widespread mechanism for inactivation of known and putative tumor suppressor genes in many types of cancers. GSTP1, CDKN2A, CDH1, APC, and RASSF1A among others are known to be downregulated in prostate cancer. The aim of this study was to search for hitherto unknown genes that are upregulated after treatment by demethylating agents (i.e. DMNT inhibitors) thereby marking them as potential new therapeutic targets and putative biomarkers in prostate carcinogenesis. Prostate cancer cell lines DU-145 and LNCaP were treated in three independent biological experiments with 100μM zebularine for ten days and monitored for cytotoxic and apoptotic side effects. Efficacy of the treatment was verified on the RNA expression level of known epigenetically regulated genes. Expression changes of total RNA from treated and untreated cells were compared using whole genome expression microarrays (Affymetrix Human Gene 1.0 ST). A number of conspicuously upregulated genes (&amp;gt;15.0-fold, presence of CpG islands, higher expression in normal prostate) were further evaluated by Q-PCR in 50 cases of paired normal and tumor tissue samples of prostate cancer patients who underwent radical prostatectomy in our clinic between 2002 and 2004. The seryl-tRNA synthetase gene SARS was discovered by us for the first time to be downregulated in 34/50 cases (66%; p = 0,04) in prostate cancer. The DNA-damage-inducible transcript 1 (DDIT1), also known as GADD45A and was downregulated in 42/50 cases (84%; p = 0,0004). The discovery of a downregulated GADD45A in our independent experiments supports the already known fact of a methylation-mediated repression of GADD45A in prostate cancer (Ramachandran et al., 2009) and its suitability as a potential therapeutic target. In addition, we found a statistically significant inverse correlation of SARS expression and raising patient Gleason scores. Based on our estimated “upregulation-rate” of the promoter hypermethylation gold standard gene for prostate cancer GSTP1 in 98% of our patient samples (48/50; p = 0,0001) we present a robust workflow for the discovery of methylation-mediated transcriptionally inactivated genes. Citation Format: Hans Krause, Odiljon Ikromov, Imad Al Kamal, Ahmed Magheli, Carsten Kempkensteffen, Kurt Miller. Pharmacological reactivation of epigenetically regulated genes in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2972. doi:10.1158/1538-7445.AM2013-2972

N′-Formyl-2-(5-nitrothiophen-2-yl)benzothiazole-6-carbohydrazide as a potential anti-tumour agent for prostate cancer in experimental studies

Benzothiazoles (BZTs) represent organic compounds with different biological actions. In this study we aimed to investigate ten newly synthesized BZT derivatives as potential anti-tumour agents against prostate cancer in vitro and in vivo. The cytotoxic effect of these compounds was screened on the human prostate cancer cell lines PC-3 and LNCaP. The most effective compound, N'-formyl-2-(5-nitrothiophen-2-yl)benzothiazole-6-carbohydrazide, was further characterized regarding its dose- and time-dependent effects on cell viability and proliferation (XTT test) as well as on adhesion and spreading (real-time cell analyzer xCelligence), migration (scratch-wound repair assay) and invasion (Boyden chamber) of the cells. This BZT derivative was also tested as an inhibitor of angiogenesis (chicken chorioallantoic membrane assay), clonogenic activity (soft agar) and matrix metalloproteinase 9 (gelatin zymography). N'-Formyl-2-(5-nitrothiophen-2-yl)benzothiazole-6-carbohydrazide significantly inhibited all tested properties of the prostate cancer cell lines and showed low toxic in vitro and in vivo effects. The in vitro anti-tumour activity of this compound was confirmed by the in vivo effects on PC-3 xenografts in nude mice. Tumour growth was decreased in treated compared with untreated mice. These results suggest the potential capacity of BZTs and in particular N'-formyl-2-(5-nitrothiophen-2-yl)benzothiazole-6-carbohydrazide as anti-tumour agents for the treatment of prostate cancer.

Abstract 3629: HOXA10 expression profiles in prostate cancer

Abstract HOXA10 is a member of the homeobox gene family and is a transcription factor primarily involved in embryonic development. Perturbed HOX gene expression has been identified in numerous cancers and thus suggests that these genes may play important roles in diagnosis and treatment of cancer. We initially identified altered HOXA10 expression in prostate tumors form prostate-specific PTEN conditional knockout mice. HOXA10 is highly expressed in the normal prostate glands from wildtype and PTEN-mutant mice and gradually decreases with tumor development and decreased differentiation. However, focal expression of HOXA10 is observed in poorly differentiated tumors and is highly expressed in metastases. To determine clinical significance of HOXA10 in human prostate cancer, we examined HOXA10 expression by immunohistochemistry in matched normal and cancer specimens from prostatectomy cases. HOXA10 nuclear expression was measured according to intensity and distribution. Our analysis shows that HOXA10 is strongly expressed in benign glands and low expression is significantly correlated with Gleason grade (p&amp;lt;0.001), tumor differentiation (p&amp;lt;0.02), pathological stage (p&amp;lt;0.001) and PSA-failure (p&amp;lt;0.001). Expression of HOXA10 was also evaluated in human prostate cell lines and was strong in the benign prostatic hyperplasia BPH-1 human prostate cell line and weak to absent in the malignant human prostate cancer cell lines PC3, DU145 and LNCaP. These data show that HOXA10 is related to tumorigenesis of prostate cancer prostate cancer and warrants further investigation into the functional role of HOXA10 in tumor development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3629. doi:1538-7445.AM2012-3629