Clara Hwang

Loss of Let-7 Up-Regulates EZH2 in Prostate Cancer Consistent with the Acquisition of Cancer Stem Cell Signatures That Are Attenuated by BR-DIM

The emergence of castrate-resistant prostate cancer (CRPC) contributes to the high mortality of patients diagnosed with prostate cancer (PCa), which in part could be attributed to the existence and the emergence of cancer stem cells (CSCs). Recent studies have shown that deregulated expression of microRNAs (miRNAs) contributes to the initiation and progression of PCa. Among several known miRNAs, let-7 family appears to play a key role in the recurrence and progression of PCa by regulating CSCs; however, the mechanism by which let-7 family contributes to PCa aggressiveness is unclear. Enhancer of Zeste homolog 2 (EZH2), a putative target of let-7 family, was demonstrated to control stem cell function. In this study, we found loss of let-7 family with corresponding over-expression of EZH2 in human PCa tissue specimens, especially in higher Gleason grade tumors. Overexpression of let-7 by transfection of let-7 precursors decreased EZH2 expression and repressed clonogenic ability and sphere-forming capacity of PCa cells, which was consistent with inhibition of EZH2 3′UTR luciferase activity. We also found that the treatment of PCa cells with BR-DIM (formulated DIM: 3,3′-diindolylmethane by Bio Response, Boulder, CO, abbreviated as BR-DIM) up-regulated let-7 and down-regulated EZH2 expression, consistent with inhibition of self-renewal and clonogenic capacity. Moreover, BR-DIM intervention in our on-going phase II clinical trial in patients prior to radical prostatectomy showed upregulation of let-7 consistent with down-regulation of EZH2 expression in PCa tissue specimens after BR-DIM intervention. These results suggest that the loss of let-7 mediated increased expression of EZH2 contributes to PCa aggressiveness, which could be attenuated by BR-DIM treatment, and thus BR-DIM is likely to have clinical impact.

IAP Antagonists Enhance Apoptotic Response to Enzalutamide in Castration-Resistant Prostate Cancer Cells via Autocrine TNF-α Signaling: IAP Antagonism in Prostate Cancer

Castration‐resistant prostate cancer (CRPC) remains incurable and identifying effective treatments continues to present a clinical challenge. Although treatment with enzalutamide, a second generation androgen receptor (AR) antagonist, prolongs survival in prostate cancer patients, responses can be limited by intrinsic resistance or acquired resistance. A potential mechanism of resistance to androgen axis inhibition is evasion of apoptosis. Inhibitor of apoptosis proteins (IAPs) are found to be overexpressed in prostate cancer and function to block apoptosis and promote survival signaling. Novel, small‐molecule IAP antagonists, such as AEG40995, are emerging as a strategy to induce apoptosis and increase therapeutic response in cancer.

Does KRAS Testing in Metastatic Colorectal Cancer Impact Overall Survival? A Comparative Effectiveness Study in a Population-Based Sample

Purpose Epidermal growth factor receptor (EGFR) inhibitors are approved for treating metastatic colorectal cancer (CRC); KRAS mutation testing is recommended prior to treatment. We conducted a non-inferiority analysis to examine whether KRAS testing has impacted survival in CRC patients. Patients and Methods We included 1186 metastatic CRC cases from seven health plans. A cutpoint of July, 2008, was used to define two KRAS testing time period groups: “pre-testing” (n = 760 cases) and “post-testing” (n = 426 cases). Overall survival (OS) was estimated, and the difference in median OS between the groups was calculated. The lower bound of the one-sided 95% confidence interval (CI) for the difference in survival was used to test the null hypothesis of post-testing inferiority. Multivariable Cox regression models were constructed to adjust for covariates. Results The median unadjusted OS was 15.4 months (95% CI: 14.0–17.5) and 12.8 months (95% CI: 10.0–15.2) in the pre- and post-testing groups, respectively. The OS difference was −2.6 months with one-sided 95% lower confidence bound of −5.13 months, which was less than the non-inferiority margin (−5.0 months, unadjusted p = 0.06), leading to a failure to reject inferiority of OS in the post-testing period. In contrast, in the adjusted analysis, OS non-inferiority was identified in the post-testing period (p = 0.001). Sensitivity analyses using cutpoints before and after July, 2008, also met the criteria for non-inferiority. Conclusion Implementation of KRAS testing did not influence CRC OS. Our data support the use of KRAS testing to guide administration of EGFR inhibitors for treatment of metastatic CRC without diminished OS.

Enrichment and mutation detection of circulating tumor cells from blood samples

The potential of circulating tumor cells (CTCs) in the diagnosis and prognosis of cancer patients has become increasingly attractive. However, molecular analysis of CTCs is hindered by low sensitivity and a high level of background leukocytes in CTC enrichment technologies. We have developed a novel protocol using a microfluidic device, which enriches and retrieves CTCs from blood samples. The principle of CTC capturing is that tumor cells are larger and less deformable than normal blood cells. To evaluate the potential of utilizing Celsee PREP100 in CTC molecular analysis, we prepared prostate cancer cell lines PC3 and LNCaP, retrieved the captured cells and analyzed them using PCR amplicon sequencing. We were able to recover an average of 79% of 110-1,100 PC3 and 60–1,500 LNCaP cells, and detect the p.K139fs*3 deletion of the p53 gene in PC3 cells and p.T877A mutation of the androgen receptor gene in LNCaP cells. Next, we spiked these two types of cells into normal donor blood samples, captured the cells and analyzed them using PCR amplicon sequencing. The PC3 and LNCaP cells were captured and retrieved with the ratio of captured CTCs to the background leukocytes reaching 1:1.5 for PC3 and 1:2.9 for LNCaP cells. We further revealed that the p.K139fs*3 deletion and p.T877A mutation can be detected in the captured PC3 and LNCaP cells, respectively. We successfully validated this approach using clinical blood samples from patients with metastatic prostate cancer. Our results demonstrated a novel approach for CTC enrichment and illustrated the potential of CTC molecular characterization for diagnosis, prognosis and treatment selection of patients with metastatic malignancy.

Abstract 3571: Targeted suppression of inhibitor of apoptosis proteins amplifies apoptosis and improves response to enzalutamide in prostate cancer

Prostate cancer is the most common malignancy and the 2nd leading cause of cancer death in men worldwide. First-line treatment includes androgen deprivation therapy in addition to androgen receptor (AR) antagonists that work to block androgen signaling. Although most patients respond initially to hormone deprivation therapy, responses are not durable and the disease progresses to a lethal stage, termed castrate resistant prostate cancer (CRPC). Interestingly, most CRPCs are still dependent on AR signaling for growth and survival, therefore more potent AR-antagonists such as enzalutamide have recently been FDA approved for treatment of CRPC. However, response rates to enzalutamide can be limited by emergence of acquired resistance or intrinsic resistance. A universal mechanism of resistance to cytotoxic therapies is upregulation of anti-apoptotic programs that function to override death signals and permit survival of the tumor cell. In prostate cancer, overexpression of the Inhibitor of Apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP has been demonstrated in progression to CRPC. Additionally, aberrant expression of these proteins is reported to contribute to anti-androgen resistance. Therefore, to achieve more robust responses to AR inhibition with enzalutamide, we targeted IAP proteins in order to amplify the apoptotic signal and increase cell death in prostate cancer cells. Using a small-molecule IAP inhibitor, we evaluated response to enzalutamide upon IAP inhibition in prostate cancer cell lines LNCAP and C4-2. Here we demonstrate greater proliferation inhibition, reduced cell survival and synergistic induction of apoptosis signaling in response to combined AR antagonism and IAP inhibition. Furthermore, we demonstrate depletion of cIAP1 protein expression upon enzalutamide treatment indicating a possible role for AR in regulating anti-apoptotic pathways. Taken together these results demonstrate that IAP proteins may be a critical survival pathway in CRPC and antagonism of these proteins through small-molecule inhibition can amplify apoptosis and increase cell death in response to enzalutamide. Targeting a critical survival pathway can lower the apoptotic threshold providing a possible therapeutic opportunity to treat and prevent resistance to enzalutamide and increase benefit to patients with this lethal form of prostate cancer. Citation Format: Amanda Pilling, Ok Hwang, Clara Hwang. Targeted suppression of inhibitor of apoptosis proteins amplifies apoptosis and improves response to enzalutamide in 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 3571.

A biomarker trial of BR-DIM (BioResponse 3,3’- Diindolylmethane) in patients with prostate cancer who undergo prostatectomy.

e15185 Background: DIM acts as an anti-androgen which down-regulates the androgen receptor (AR) and prostate specific antigen (PSA). We conducted a biomarker trial to measure the level of DIM and its effects on AR in prostate tissue after treatment of prostate cancer (PCa) patients with BR-DIM. METHODS Patients with clinically confined PCa who were candidates for surgery were treated with BR-DIM at a dose of 225 mg orally twice daily for a minimum of 14 days. Patients did not receive any prior therapy for prostate cancer and had adequate organ function. Men received their last dose of BR-DIM the day before surgery. Blood samples for DIM levels were collected at pre-treatment and at ~12 h after the last dose of BR-DIM (just prior to the surgery). The prostate was removed during surgery and DIM concentration was measured in the prostate tissue. AR was evaluated by immunohistochemistry. RESULTS 21 patients were treated at 2 institutions. The 20 evaluable patients had median age 60 (range 41 - 76), 10 were Caucasian, and 10 were African American. Toxicity was minimal; only two patients with grade 3 headache. DIM was found at a mean trough level of 10 ng/ml (range, < 1 to 23) and 15 ng/g (range, < 1 to 26.8) in plasma and prostate tissue, respectively. PSA remained the same pre and post BR-DIM treatment. In our interim analysis for AR expression, 20 PCa control specimens (non-intervention samples) and 8 patients tumors from BR-DIM intervention samples, and two cases having pre-DIM diagnostic biopsy and subsequent post BR-DIM samples were evaluated. Strong AR expression was found in 9 of 20 randomly selected PCa specimens (45%) and others showed weak or no positive staining. Of the 8 PCa specimens after BR-DIM intervention, AR staining was seen in 3 cases (37.5%). These results suggest that BR-DIM intervention slightly down-regulated AR expression, consistent with pre-clinical findings. CONCLUSIONS BR-DIM was well tolerated by our 21 patients. DIM was detected in both plasma and prostate tissues at ~12 h post dosing. AR expression was lost in 5 of 8 patients after BR-DIM treatment. Additional studies using BR-DIM in PCa are warranted.