Kai Lee Yap

NAC1 modulates sensitivity of ovarian cancer cells to cisplatin by altering the HMGB1-mediated autophagic response

Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, is known to have important roles in proliferation and growth of tumor cells and in chemotherapy resistance. Yet, the mechanisms underlying how NAC1 contributes to drug resistance remain largely unclear. We report here that autophagy was involved in NAC1-mediated resistance to cisplatin, a commonly used chemotherapeutic drug in the treatment of ovarian cancer. We found that treatment with cisplatin caused an activation of autophagy in ovarian cancer cell lines, A2780, OVCAR3 and SKOV3. We further demonstrated that knockdown of NAC1 by RNA interference or inactivation of NAC1 by inducing the expression of a NAC1 deletion mutant that contains only the BTB/POZ domain significantly inhibited the cisplatin-induced autophagy, resulting in increased cisplatin cytotoxicity. Moreover, inhibition of autophagy and sensitization to cisplatin by NAC1 knockdown or inactivation were accompanied by induction of apoptosis. To confirm that the sensitizing effect of NAC1 inhibition on the cytotoxicity of cisplatin was attributed to suppression of autophagy, we assessed the effects of the autophagy inhibitors 3-methyladenosine and chloroquine, and small interfering RNAs (siRNAs) targeting beclin 1 or Atg5 on the cytotoxicity of cisplatin. Treatment with 3-methyladenosine, chloroquine or beclin 1 and Atg5-targeted siRNA also enhanced the sensitivity of SKOV3, A2780 and OVCAR3 cells to cisplatin, indicating that suppression of autophagy indeed renders tumor cells more sensitive to cisplatin. Regulation of autophagy by NAC1 was mediated by the high-mobility group box 1 (HMGB1), as the functional status of NAC1 was associated with the expression, translocation and release of HMGB1. The results of our study not only revealed a new mechanism determining cisplatin sensitivity but also identified NAC1 as a novel regulator of autophagy. Thus, the NAC1-mediated autophagy may be exploited as a new target for enhancing the efficacy of cisplatin against ovarian cancer and other types of malignancies.

NAC-1, a potential stem cell pluripotency factor, contributes to paclitaxel resistance in ovarian cancer through inactivating Gadd45 pathway.

Nucleus accumbens-1 (Nac1 or NAC-1) belongs to the BTB/POZ (Pox virus and Zinc finger/Bric-a-brac Tramtrack Broad complex) transcription factor family and is a novel protein that potentially participates in self-renewal and pluripotency in embryonic stem cells. In human cancer, NAC-1 is upregulated in several types of neoplasms, but particularly in recurrent chemoresistant ovarian carcinomas, suggesting a biological role for NAC-1 in the development of drug resistance in ovarian cancer. We have assessed this possibility and shown a correlation between NAC-1 expression and ex vivo paclitaxel resistance in ovarian serous carcinoma tissues and cell lines. We found that expression of Gadd45-γ-interacting protein 1 (Gadd45gip1), a downstream target negatively regulated by NAC-1, was reduced in paclitaxel-resistant cells. Ectopic expression of NAC-1 or knockdown of Gadd45gip1 conferred paclitaxel resistance, whereas NAC-1 knockdown or ectopic expression of Gadd45gip1 increased paclitaxel sensitivity. Furthermore, silencing NAC-1 expression or disrupting NAC-1 homodimerization by a dominant negative NAC-1 protein that contained only the BTB/POZ domain induced the expression of Gadd45γ, which interacted with Gadd45gip1. Reducing Gadd45γ expression by small hairpin RNAs partially enhanced paclitaxel resistance. Thus, this study provides new evidence that NAC-1 upregulation and homodimerization contribute to tumor recurrence by equipping ovarian cancer cells with the paclitaxel-resistant phenotype through negative regulation of the Gadd45 pathway.

Afatinib Activity in Platinum-Refractory Metastatic Urothelial Carcinoma in Patients With ERBB Alterations

PURPOSE Somatic mutations and copy number variation in the ERBB family are frequent in urothelial carcinoma (UC) and may represent viable therapeutic targets. We studied whether afatinib (an oral, irreversible inhibitor of the ErbB family) has activity in UC and if specific ERBB molecular alterations are associated with clinical response. PATIENTS AND METHODS In this phase II trial, patients with metastatic platinum-refractory UC received afatinib 40 mg/day continuously until progression or intolerance. The primary end point was 3-month progression-free survival (PFS3). Prespecified tumor analysis for alterations in EGFR, HER2, ERBB3, and ERBB4 was conducted. RESULTS The first-stage enrollment goal of 23 patients was met. Patient demographic data included: 78% male, median age 67 years (range, 36 to 82 years), hemoglobin < 10 g/dL in 17%, liver metastases in 30%, median time from prior chemotherapy of 3.6 months, and Eastern Cooperative Oncology Group performance status ≤ 1 in 100%. No unexpected toxicities were observed; two patients required dose reduction for grade 3 fatigue and rash. Overall, five of 23 patients (21.7%) met PFS3 (two partial response, three stable disease). Notably, among the 21 tumors analyzed, five of six patients (83.3%) with HER2 and/or ERBB3 alterations achieved PFS3 (PFS = 10.3, 7.0, 6.9, 6.3, and 5.0 months, respectively) versus none of 15 patients without alterations (P < .001). Three of four patients with HER2 amplification and three of three patients with ERBB3 somatic mutations (G284R, V104M, and R103G) met PFS3. One patient with both HER2 amplification and ERBB3 mutation never progressed on therapy, but treatment was discontinued after 10.3 months as a result of depressed ejection fraction. The median time to progression/discontinuation was 6.6 months in patients with HER2/ERBB3 alterations versus 1.4 months in patients without alterations (P < .001). CONCLUSION Afatinib demonstrated significant activity in patients with platinum-refractory UC with HER2 or ERBB3 alterations. The potential contribution of ERBB3 to afatinib sensitivity is novel. Afatinib deserves further investigation in molecularly selected UC.

Oncoproteomic Analysis Reveals Co-Upregulation of RELA and STAT5 in Carboplatin Resistant Ovarian Carcinoma

Background Ovarian cancer is one of the most lethal types of female malignancy. Although most patients are initially responsive to platinum-based chemotherapy, almost all develop recurrent chemoresistant tumors and succumb to their diseases. Elucidating the pathogenesis underlying drug resistance is fundamental to the development of new therapeutics, leading to improved clinical outcomes in these patients. Methods and Findings We compared the proteomes of paired primary and recurrent post-chemotherapy ovarian high-grade serous carcinomas from nine ovarian cancer patients using CIEF/Nano-RPLC coupled with ESI-Tandem MS. As compared to their primary tumors, more than half of the recurrent tumors expressed higher levels of several proteins including CP, FN1, SYK, CD97, AIF1, WNK1, SERPINA3, APOD, URP2, STAT5B and RELA (NF-κB p65), which were also validated by quantitative RT-PCR. Based on shRNA screening for the upregulated genes in in vitro carboplatin-resistant cells, we found that simultaneous knockdown of RELA and STAT5B was most effective in sensitizing tumor cells for carboplatin treatment. Similarly, the NF-κB inhibitor, BMS-345541, and the STAT5 inhibitor, Dasatinib, significantly enhanced cell sensitivity to carboplatin. Moreover, both RELA and STAT5 are known to bind to the promoter region of Bcl-X, regulating its promoter activity. In this regard, augmented Bcl-xL expression was detected in carboplatin-resistant cells. Combined ectopic expression of RELA and STAT5B enhanced Bcl-xL promoter activity while treatment with BMS-345541 and Dasatinib decreased it. Chromatin immunoprecipitation of the Bcl-X promoter region using a STAT5 antibody showed induction of RELA and STAT5 DNA-binding segments both in naïve cells treated with a high concentration of carboplatin as well as in carboplatin-resistant cells. Conclusions Proteomic analysis identified RELA and STAT5 as two major proteins associated with carboplatin resistance in ovarian tumors. Our results further showed that NF-κB and STAT5 inhibitor could sensitize carboplatin-resistant cells and suggest that such inhibitors can be used to benefit patients with carboplatin-resistant recurrent ovarian cancer.

Expression of Fatty Acid Synthase Depends on NAC1 and Is Associated with Recurrent Ovarian Serous Carcinomas.

Our previous reports demonstrated that NAC1, a BTB/POZ domain-containing nuclear protein, upregulates in recurrent ovarian serous carcinoma and participates in developing drug resistance in cancer cells. The current study applies quantitative proteomics to identify the proteins controlled by NAC1 by comparing the proteomes of SKOV3 cells with and without expression of a dominant negative NAC1 construct, N130. From the proteins that are downregulated by N130 (upregulated by NAC1), we chose to further characterize fatty acid synthase (FASN). Similar to change in protein level, the FASN transcript level in SKOV3 cells was significantly reduced by N130 induction or by NAC1 knockdown. Immunohistochemistry showed that NAC1 and FASN immunointensities in ovarian serous carcinoma tissues had a highly significant correlation (P < .0001). Moreover, we found that recurrent serous carcinomas exhibited higher FASN immunointensities than their matched primary tumors (P < .001). Multivariate analysis showed that an FASN staining score of >1 in serous carcinomas was associated with a worse overall survival time (P < .01). Finally, C93, a new FASN inhibitor, induced massive apoptosis in carboplatin/paclitaxel resistant ovarian cancer cells. In conclusion, we show that NAC1 is essential for FASN expression in ovarian serous carcinomas and the expression of FASN significantly correlates with tumor recurrence and disease aggressiveness. The dependence of drug resistant tumor cells on FASN suggests a potential application of FASN-based therapeutics for recurrent ovarian cancer patients.

Whole-Exome Sequencing of Muscle-Invasive Bladder Cancer Identifies Recurrent Mutations of UNC5C and Prognostic Importance of DNA Repair Gene Mutations on Survival

Purpose: Because of suboptimal outcomes in muscle-invasive bladder cancer even with multimodality therapy, determination of potential genetic drivers offers the possibility of improving therapeutic approaches and discovering novel prognostic indicators. Experimental Design: Using pTN staging, we case-matched 81 patients with resected ≥pT2 bladder cancers for whom perioperative chemotherapy use and disease recurrence status were known. Whole-exome sequencing was conducted in 43 cases to identify recurrent somatic mutations and targeted sequencing of 10 genes selected from the initial screening in an additional 38 cases was completed. Mutational profiles along with clinicopathologic information were correlated with recurrence-free survival (RFS) in the patients. Results: We identified recurrent novel somatic mutations in the gene UNC5C (9.9%), in addition to TP53 (40.7%), KDM6A (21.0%), and TSC1 (12.3%). Patients who were carriers of somatic mutations in DNA repair genes (one or more of ATM, ERCC2, FANCD2, PALB2, BRCA1, or BRCA2) had a higher overall number of somatic mutations (P = 0.011). Importantly, after a median follow-up of 40.4 months, carriers of somatic mutations (n = 25) in any of these six DNA repair genes had significantly enhanced RFS compared with noncarriers [median, 32.4 vs. 14.8 months; hazard ratio of 0.46, 95% confidence interval (CI), 0.22–0.98; P = 0.0435], after adjustment for pathologic pTN staging and independent of adjuvant chemotherapy usage. Conclusion: Better prognostic outcomes of individuals carrying somatic mutations in DNA repair genes suggest these mutations as favorable prognostic events in muscle-invasive bladder cancer. Additional mechanistic investigation into the previously undiscovered role of UNC5C in bladder cancer is warranted. Clin Cancer Res; 20(24); 6605–17. ©2014 AACR.

Quantitative characterization of T-cell repertoire in allogeneic hematopoietic stem cell transplant recipients

Allogeneic hematopoietic stem cell transplantation (HSCT) is one of curative treatment options for patients with hematologic malignancies. Although GVHD mediated by the donor’s T lymphocytes remains the most challenging toxicity of allo-HSCT, graft-versus-leukemia (GVL) effect targeting leukemic cells, has an important role in affecting the overall outcome of patients with AML. Here we comprehensively characterized the TCR repertoire in patients who underwent matched donor or haplo-cord HSCT using next-generation sequencing approach. Our study defines the functional kinetics of each TCRA and TCRB clone, and changes in T-cell diversity (with identification of CDR3 sequences) and the extent of clonal expansion of certain T-cells. Using this approach, our study demonstrates that higher percentage of cord-blood cells at 30 days after transplant was correlated with higher diversity of TCR repertoire, implicating the role of cord-chimerism in enhancing immune recovery. Importantly, we found that GVHD and relapse, exclusive of each other, were correlated with lower TCR repertoire diversity and expansion of certain T-cell clones. Our results highlight novel insights into the balance between GVHD and GVL effect, suggesting that higher diversity early after transplant possibly implies lower risks of both GVHD and relapse following the HSCT transplantation.

Low T-cell Receptor Diversity, High Somatic Mutation Burden, and High Neoantigen Load as Predictors of Clinical Outcome in Muscle-invasive Bladder Cancer

BACKGROUND The success of cancer immunotherapies has highlighted the potent ability of local adaptive immune responses to eradicate cancer cells by targeting neoantigens generated by somatic alterations. However, how these factors interact to drive the natural history of muscle-invasive bladder cancer (MIBC) is not well understood. OBJECTIVE To investigate the role of immune regulation in MIBC disease progression, we performed massively parallel T-cell receptor (TCR) sequencing of tumor-infiltrating T cells (TILs), in silico neoantigen prediction from exome sequences, and expression analysis of immune-related genes. DESIGN, SETTING, AND PARTICIPANTS We analyzed 38 MIBC tissues from patients who underwent definitive surgery with a minimum clinical follow-up of 2 yr. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Recurrence-free survival (RFS) was determined. TCR diversity was quantified using Simpsons diversity index. The main analyses involved the Mann-Whitney U test, Kaplan-Meier survival analysis, and Cox proportional hazards models. RESULTS AND LIMITATIONS Low TCRβ chain diversity, correlating with oligoclonal TIL expansion, was significantly correlated with longer RFS, even after adjustment for pathologic tumor stage, node status, and receipt of adjuvant chemotherapy (hazard ratio 2.67, 95% confidence interval 1.08-6.60; p=0.03). Patients with both a high number of neoantigens and low TCRβ diversity had longer RFS compared to those with fewer neoantigens and high TCR diversity (median RFS 275 vs 30 wk; p=0.03). Higher expression of immune cytolytic genes was associated with nonrecurrence among patients with low TCR diversity or fewer neoantigens. Limitations include the sample size and the inability to distinguish CD8+ and CD4+ T cells using TCR sequencing. CONCLUSIONS These findings are the first to show that detailed tumor immune-genome analysis at definitive surgery can identify molecular patterns of antitumor immune response contributing to better clinical outcomes in MIBC. PATIENT SUMMARY We discovered that clonal expansion of certain T cells in tumor tissue, possibly targeting cancer-specific antigens, contributes to prevention of bladder cancer recurrence.

Dysfunction of Nucleus Accumbens-1 Activates Cellular Senescence and Inhibits Tumor Cell Proliferation and Oncogenesis

Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, has emerging roles in cancer. We report here that NAC1 acts as a negative regulator of cellular senescence in transformed and nontransformed cells, and dysfunction of NAC1 induces senescence and inhibits its oncogenic potential. We show that NAC1 deficiency markedly activates senescence and inhibits proliferation in tumor cells treated with sublethal doses of γ-irradiation. In mouse embryonic fibroblasts from NAC1 knockout mice, following infection with a Ras virus, NAC1-/- cells undergo significantly more senescence and are either nontransformed or less transformed in vitro and less tumorigenic in vivo when compared with NAC1+/+ cells. Furthermore, we show that the NAC1-caused senescence blunting is mediated by ΔNp63, which exerts its effect on senescence through p21, and that NAC1 activates transcription of ΔNp63 under stressful conditions. Our results not only reveal a previously unrecognized function of NAC1, the molecular pathway involved and its impact on pathogenesis of tumor initiation and development, but also identify a novel senescence regulator that may be exploited as a potential target for cancer prevention and treatment.

NAC1 Is an Actin-Binding Protein That Is Essential for Effective Cytokinesis in Cancer Cells

NAC1 is a transcriptional corepressor protein that is essential to sustain cancer cell proliferation and migration. However, the underlying molecular mechanisms of NAC1 function in cancer cells remain unknown. In this study, we show that NAC1 functions as an actin monomer-binding protein. The conserved BTB protein interaction domain in NAC1 is the minimal region for actin binding. Disrupting NAC1 complex function by dominant-negative or siRNA strategies reduced cell retraction and abscission during late-stage cytokinesis, causing multinucleation in cancer cells. In Nac1-deficient murine fibroblasts, restoring NAC1 expression was sufficient to partially avert multinucleation. We found that siRNA-mediated silencing of the actin-binding protein profilin-1 in cancer cells caused a similar multinucleation phenotype and that NAC1 modulated the binding of actin to profillin-1. Taken together, our results indicate that the NAC1/actin/profilin-1 complex is crucial for cancer cell cytokinesis, with a variety of important biologic and clinical implications.