Racial Disparity Among Women Diagnosed With Invasive Breast Cancer in a Large Integrated Health System

Purpose: Reasons for the well-described disparity in outcomes between African American (AA) and non-Hispanic white (NHW) women with invasive breast cancer are unclear, making it difficult to identify solutions. This study examined the effects of demographics, biomarkers, tumor characteristics, cancer stage, morphology, and treatment variables on overall and cancer-free survival in these patient populations. Methods: We retrospectively reviewed data for 6231 patients diagnosed with invasive breast cancer throughout an integrated health system from January 2006 through March 2015. Included for analysis were 5023 NHW and 413 AA women. All category and continuous variables in the study were described in the two groups using appropriate statistics. Kaplan-Meier method of survival with log-rank test was used to compare the two racial groups (NHW and AA). Cox proportional hazards regression was used to find hazard ratios for the predictors of survival and recurrence-free survival probability. Propensity probability match method (1:1) was used to match 319 NSW women to 319 similar AA women. Matching was done using all significant predictors, including demographic variables. Results: Compared to NHW women, AA women presented with invasive breast cancer at a younger age (P < 0.001) and had a higher proportion of stage IV cancers (P < 0.001), which were more often infiltrating ductal carcinoma (P < 0.003) and poorly differentiated (P < 0.001). Within 10-year follow-up, AA women had shorter overall and recurrence-free survival (log-rank P < 0.001), were 1.4 times more likely to die (P = 0.009), and were twice as likely to have recurrence (P < 0.001) than NHW women. In the matched groups, overall survival was similar for AA and NHW (log-rank P = 0.0793); however, recurrence-free survival was higher in NHW than in AA women (P = 0.047). Conclusions: When presenting characteristics of AA and NHW women with invasive breast cancer are matched, disparity in overall mortality and rate of recurrence appears to be reduced or perhaps eliminated, suggesting invasive breast cancers in AA and NHW women respond similarly to treatment. Further study is needed to explore the true effect of biological factors; however, rectifying delivery of and access to care might be expected to mitigate, in large part, the racial disparity currently seen in breast cancer outcomes

P53 level and MGMT promoter methylation are playing a role in Zika virus replication in glioblastoma

Background: Glioblastoma (GBM) is a malignant primary brain cancer. The poor median survival rate for patients with GBM of 15 months has not budged for the past 15 years, when the current standard treatment was first approved. There is no standard-of-care chemotherapy for recurrent GBM. Needless to say, novel treatments and treatment strategies for GBM are needed. One such novel treatment strategy is an oncolytic virus. The Zika virus’ (ZIKV) affinity for fetal neural stem cells has made it a compelling candidate as an oncolytic therapy for GBM. Previous studies have shown that ZIKV does infect and replicate in most but not all GBM cancer cells. Understanding the genetic milieu that is permissive for ZIKV infection is critical to the creation of a safe and effective viral oncolytic treatment. This report presents initial data for a ZIKV replication gene signature. Purpose: We hypothesized that p53 level and MGMT promoter methylation are playing a role in ZIKV replication in GBM cancer cells. Methods: ZIKV strain MR766 was propagated in Vero cells. Viral stock was titrated by plaque assays. Western blot was used to characterize MGMT, AXL, p53, and Sox2 expression in 8 commercial GBM cell lines (LN229, U87, A172, U251, LN18, T98G, U137, and U118). The cell lines were stratified by MGMT promoter methylation status and were exposed to ZIKV at a multiplicity of infection of 1. The percentage of infected cells was quantified by flow cytometry using the pan-flavivirus anti-E protein Ab 4G2. We also compared RNA-seq data of ZIKV replicating and nonreplicating GBM cell lines to find differentially expressed genes. Results: In this study, we found that AXL was overexpressed in all 8 commercial GBM cell lines. With flow cytometry, we found that productive ZIKV replication only occurs in the MGMT-methylated (LN229, A172, U87, and U251) cell lines and not in MGMTunmethylated (T98G, LN18, U118, and U138) cell lines. qRT-PCR data showed that ZIKV enters all 8 cell lines but can only replicate in MGMT-methylated cell lines. Additionally, p53 expression trended lower in MGMT-methylated cell lines. RNA sequencing data comparing ZIKV replicating and nonreplicating GBM lines has identified multiple differentially genes. Conclusion: Based on these results, there is a clear difference in the ability of the Zika virus to replicate in glioblastoma cell lines based on MGMT and p53 expression. Additional work is underway to understand the mechanism(s) underlying these findings and to define a ZIKV replication gene signature

Blockade Of Mgmt Expression By O\u3csup\u3e6\u3c/sup\u3e Benzyl Guanine Leads To Inhibition Of Pancreatic Cancer Growth And Induction Of Apoptosis

Purpose: We sought to determine whether administration of a MGMT blocker, O6-benzyl guanine (O6BG), at an optimal biological dose alone or in combination with gemcitabine inhibits human pancreatic cancer cell growth. Experimental Design: Human pancreatic cancer L3.6pl and PANC1 cells were treated with O6BG, either alone or in combination with gemcitabine, and the therapeutic efficacy and biological activity of these drug combinations were investigated. Results: O6BG sensitized pancreatic cancer cells to gemcitabine. Protein and mRNA expression of MGMT, cyclin B1, cyclin B2, cyclin A, and ki-67 were significantly decreased in the presence of O 6BG. In sharp contrast, protein expression and mRNA message of p21cip1 were significantly increased. Interestingly, O6BG increases p53-mediated p21cip1 transcriptional activity and suppresses cyclin B1. In addition, our results indicate that p53 is recruited to p21 promoter. Furthermore, an increase in p21cip1 and a decrease in cyclin transcription are p53 dependent. The volume of pancreatic tumors was reduced by 27% in mice treated with gemcitabine alone, by 47% in those treated with O6BG alone, and by 65% in those mice given combination. Immunohistochemical analysis showed that O6BG inhibited expression of MGMT and cyclins, and increased expression of p21cip1. Furthermore, there was a significant decrease in tumor cell proliferation and an increase in tumor cell apoptosis. Conclusions: Collectively, our results show that decreased MGMT expression is correlated with p53 activation, and significantly reduced primary pancreatic tumor growth. These findings suggest that O6BG either alone or in combination with gemcitabine may provide a novel and effective approach for the treatment of human pancreatic cancer. © 2009 American Association for Cancer Research

Zika virus targets stem cell markers and modulates antiviral responses in glioblastoma cancer stem cells

Background: Glioblastoma is the most common and lethal form of brain cancer. Standard treatment involves surgery, chemotherapy, and radiation. Tumor recurrence is caused by a population of glioblastoma cancer stem cells (GSCs) that resist and survive treatment. There are currently no pharmacological agents available for specific targeting of GSCs. Zika virus (ZIKV) is a flavivirus that targets normal neural stem cells in the developing brain and causes microcephaly. ZIKV also selectively targets GSCs in a similar manner. We examined the effect of ZIKV on specific stem cell markers as well as antiviral responses in patient-derived GSCs. Purpose: To understand the oncolytic mechanism of ZIKV toward GSCs. Methods: Glioblastoma patient-derived cell lines were acquired from within our institution and used for the entirety of this study. Patient-derived cell lines were grown in spheres using supplemented neurocult media. ZIKV strain MR766 was propagated in Vero cells, and viral stock was titrated by plaque assays. Glioblastoma patient cell lines were infected with ZIKV at a multiplicity of infection of 1. The percentage of infection was quantified by flow cytometry using a pan-flavivirus antibody. Western blotting was used to characterize protein expression, while qRT-PCR was used to quantify gene expression pre- and post-ZIKV infection in patient-derived cell lines. Results: We tested multiple stem cell markers and found that Sox2 and Nestin expression is highly upregulated in our glioblastoma patient-derived cell lines (7714, 7730, 7753), while Nanog, Oct-4, and Musashi-1 were at basal levels. We further found that both Sox2 and Nestin were significantly decreased post-ZIKV infection. Next, we found that ZIKV induces antiviral responses postinfection through an increase of interferon-induced genes IRF1, IFIT1, ISG15, and IL6. Lastly, we found increased expression of FGF2 and Caspase-3 post-ZIKV infection, which are involved in cell differentiation and cell death, respectively. Conclusion: Our results suggest that Zika virus infection causes loss of glioblastoma stem cell self-renewal through decreased Sox2 and Nestin expression. Furthermore, ZIKV leads to antiviral responses in GSCs through an increase of multiple interferon-induced genes. Finally, genes involved in cell differentiation and cell death are upregulated in GSCs post-ZIKV infection. Thus, there are multiple mechanisms to explain GSC death following ZIKV infection

Racial Disparity Among Women Diagnosed With Invasive Breast Cancer in a Large Integrated Health System

Purpose: Reasons for the well-described disparity in outcomes between African American (AA) and non-Hispanic white (NHW) women with invasive breast cancer are unclear, making it difficult to identify solutions. This study examined the effects of demographics, biomarkers, tumor characteristics, cancer stage, morphology, and treatment variables on overall and cancer-free survival in these patient populations. Methods: We retrospectively reviewed data for 6231 patients diagnosed with invasive breast cancer throughout an integrated health system from January 2006 through March 2015. Included for analysis were 5023 NHW and 413 AA women. All category and continuous variables in the study were described in the two groups using appropriate statistics. Kaplan-Meier method of survival with log-rank test was used to compare the two racial groups (NHW and AA). Cox proportional hazards regression was used to find hazard ratios for the predictors of survival and recurrence-free survival probability. Propensity probability match method (1:1) was used to match 319 NSW women to 319 similar AA women. Matching was done using all significant predictors, including demographic variables. Results: Compared to NHW women, AA women presented with invasive breast cancer at a younger age (P \u3c 0.001) and had a higher proportion of stage IV cancers (P \u3c 0.001), which were more often infiltrating ductal carcinoma (P \u3c 0.003) and poorly differentiated (P \u3c 0.001). Within 10-year follow-up, AA women had shorter overall and recurrence-free survival (log-rank P \u3c 0.001), were 1.4 times more likely to die (P = 0.009), and were twice as likely to have recurrence (P \u3c 0.001) than NHW women. In the matched groups, overall survival was similar for AA and NHW (log-rank P = 0.0793); however, recurrence-free survival was higher in NHW than in AA women (P = 0.047). Conclusions: When presenting characteristics of AA and NHW women with invasive breast cancer are matched, disparity in overall mortality and rate of recurrence appears to be reduced or perhaps eliminated, suggesting invasive breast cancers in AA and NHW women respond similarly to treatment. Further study is needed to explore the true effect of biological factors; however, rectifying delivery of and access to care might be expected to mitigate, in large part, the racial disparity currently seen in breast cancer outcomes

In Vitro Growth Suppression of Renal Carcinoma Cells by Curcumin

Purpose Malignant clear cell renal carcinoma (ccRCC) is an aggressive tumor highly resistant to chemotherapy and radiation. Current therapeutic approaches to management of ccRCC have not significantly improved patient survival, therefore novel therapies are needed. Activated NFκB and STAT3 expression is associated with ccRCC pathogenesis. The dietary polyphenol curcumin is a well-documented antitumor agent and a known inhibitor of NFκB and STAT3 activation. Given the lack of effective therapies that block ccRCC progression, our objective was to examine whether curcumin could suppress the growth and migration of ccRCC cells, and whether this suppression was mediated via inhibition of NFκB and STAT3 activity. Methods Human ccRCC cell lines (769-p, 786-o, Caki-1, ACHN and A-498 cells) were exposed to curcumin to assess the impact of curcumin on ccRCC cell viability. Colony formation assay was used to assess the effect of curcumin on ccRCC cell renewal capability. Effect of curcumin on apoptosis was determined by annexin V binding and mitochondrial membrane depolarization assays. The anti-migratory effect of curcumin on ccRCC cells was assessed using the wound healing assay. Effect of curcumin on NFκB and STAT3 phosphorylation in 769-p cells was determined by western blot analysis. Results In ccRCC cells, curcumin decreased cell proliferation and cell viability, abolished clonogenic property, induced apoptosis and blocked cellular migration. The growth suppressive and pro-apoptotic effects of curcumin were accompanied by decreased phosphorylation of NFκB and STAT3. Conclusions The ability of curcumin to induce apoptosis and inhibit migration of ccRCC cells justifies additional mechanistic and preclinical studies that examine the effect of curcumin or other NFκB and STAT3 inhibitors as potential suppressors of ccRCC tumorigenesis

In Vitro Growth Suppression of Renal Carcinoma Cells by Curcumin

Purpose: Malignant clear cell renal carcinoma (ccRCC) is an aggressive tumor highly resistant to chemotherapy and radiation. Current therapeutic approaches to management of ccRCC have not significantly improved patient survival, therefore novel therapies are needed. Activated NFκB and STAT3 expression is associated with ccRCC pathogenesis. The dietary polyphenol curcumin is a well-documented antitumor agent and a known inhibitor of NFκB and STAT3 activation. Given the lack of effective therapies that block ccRCC progression, our objective was to examine whether curcumin could suppress the growth and migration of ccRCC cells, and whether this suppression was mediated via inhibition of NFκB and STAT3 activity. Methods: Human ccRCC cell lines (769-p, 786-o, Caki-1, ACHN and A-498 cells) were exposed to curcumin to assess the impact of curcumin on ccRCC cell viability. Colony formation assay was used to assess the effect of curcumin on ccRCC cell renewal capability. Effect of curcumin on apoptosis was determined by annexin V binding and mitochondrial membrane depolarization assays. The anti-migratory effect of curcumin on ccRCC cells was assessed using the wound healing assay. Effect of curcumin on NFκB and STAT3 phosphorylation in 769-p cells was determined by western blot analysis. Results: In ccRCC cells, curcumin decreased cell proliferation and cell viability, abolished clonogenic property, induced apoptosis and blocked cellular migration. The growth suppressive and pro-apoptotic effects of curcumin were accompanied by decreased phosphorylation of NFκB and STAT3. Conclusions: The ability of curcumin to induce apoptosis and inhibit migration of ccRCC cells justifies additional mechanistic and preclinical studies that examine the effect of curcumin or other NFκB and STAT3 inhibitors as potential suppressors of ccRCC tumorigenesis

Generation of a Patient-Derived Brain Metastasis Breast Cancer Cell Line via Novel Orthotopic Injection Placement and Serial Mouse Transplantation to Develop PDX Mouse Model

Background: The incidence of brain metastasis appears to be increasing, potentially due to advanced technology that aids early diagnosis. Patient-derived xenografts (PDX) have high translational value, as these models retain key functional characteristics of the patient tumor. PDX models are useful to understand the molecular basis of tumorigenesis and to identify new treatment targets. However, generating a first-line PDX model is challenging as engraftment failure is high. Serial transplanting tumor tissue via mouse-to-mouse propagation increases engraftment rates and decreases PDX development time. Herein we report methods to generate a PDX cell line from patient-derived tumor tissue that includes the cerebral aqueduct as a novel intracranial orthotopic implantation site. Purpose: Develop human tumors in mouse models for therapeutic purpose. Methods: Patient-derived brain metastasis tumor tissue was enzymatically dissociated into a single cell suspension and maintained in neurocult media supplemented with human recombinant bFGF and EGF (20 ng/ml). The cells were seeded at a density of 1.0 × 104/cm2 on ultra-low attachment plates and maintained at 37°C with 5% CO2. PDX models were generated via orthotopic stereotactic surgeries. Athymic nude mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). The cerebral aqueduct was located using these coordinates from bregma: A: -5; L: +0.2; V: -2.4. Mice were injected with 5.0 × 104 cells in 2 μl of media at a rate of 0.4 ul/min. Mice were monitored daily for symptoms of tumor formation. Upon becoming symptomatic, mice were euthanized and tumor tissue was harvested for both culture and H&E stain for tumor verification. Results: Mice injected with primary patient cells (first-generation mice) developed tumors at 7 weeks (average: 6.77 weeks), second-generation mice yielded tumors at 2 weeks (average: 13.5 days), and third-generation mice replicated results from second-generation mice (average: 13 days). H&E stain revealed invasive tumor masses in the ventricular system that extended from the cerebral aqueduct to the lateral ventricles. Immunohistochemistry analysis confirmed the third-generation cell line retained key characteristics of the patient tumor. Conclusion: These methods successfully generated a PDX cell line from patient-derived brain metastasis that demonstrates reliable tumor formation and phenotypic stability. Importantly, our unique intracranial implantation site revealed several distinct masses, a hallmark of brain metastasis in patients

MGMT inhibition suppresses survivin expression in pancreatic cancer

OBJECTIVES: Survivin, an antiapoptotic gene inhibited by p53, is overexpressed in human cancers and correlates with chemotherapy resistance. Here, we investigated the mutual regulatory mechanism between MGMT (O-methylguanine DNA methyltransferase) and survivin. METHODS: This study used standard techniques for protein and messenger RNA levels, promoter activity, protein-DNA interaction, cell viability, and correlative animal model. RESULTS: O-benzylguanine (BG), a potent inhibitor of MGMT (a DNA repair protein), curtails the expression of survivin in pancreatic cancer. Silencing MGMT by small interfering RNA down-regulates survivin transcription. p53 inhibition enhances MGMT and survivin expressions. When p53 was silenced, BG-induced MGMT inhibition was not associated with the down-regulation of survivin, underscoring the regulatory role of p53 in the MGMT-survivin axis. O-benzylguanine inhibits survivin and PCNA (proliferating cell nuclear antigen) at messenger RNA and protein levels in PANC-1 and L3.6pl cells and decreases survivin promoter activity via increased p53 recruitment to the survivin promoter. In orthotopic pancreatic xenografts established in nude mice, BG ± gemcitabine (GEM) decrease survivin expression in tumor tissue; protein levels and immunohistochemistry show significant decrease in survivin and PCNA levels, which correlate with increased sensitivity to GEM. CONCLUSIONS: MGMT inhibition is associated with decrease in survivin expression and increase in sensitivity to GEM in pancreatic cancer