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Introduction: As the incidence of human papillomavirus-positive (HPV +) oropharyngeal squamous cell carcinoma (OPSCC) continues to increase, it is important to optimize treatment regimens. There is growing evidence demonstrating the efficacy and safety of anti-PD-1 immunotherapy, either as monotherapy or in combination with chemotherapy, for patients with programmed death-ligand 1 (PD-L1) positive head and neck squamous cell carcinoma. Greater PD-L1 expression has been associated with greater treatment response and improved overall survival independent of treatment modality. Prior studies have observed increased PD-L1 expression and improved treatment response in HPV(+) as compared to HPV(-) tumors. In contrast, HPV(-) disease, in which smoking is causative in most cases, is typically associated with unfavorable outcomes and lower rates of PD-L1 expression. In the present study, we sought to explore the effects of smoking on PD-L1 expression in HPV(+) tumors based on smoking status. We hypothesized that smokers would demonstrate lower levels of PD-L1 expression as compared to non-smokers. Methods: Using an existing tissue bank, we identified a cohort of 52 HPV(+) tumors from patients with available smoking data. A tissue microarray was constructed with cores taken from the paraffin embedded tumor blocks. A single 0.5 micron slide was created and we examined PDL-1 expression by immunohistochemistry in 47 intact cores. Visiopharm software was used for quantitative analysis of area stained per total cellular area (Figure 2). Mann-Whitney U test was then performed to compare PD-L1 expression between current smokers and former/never smokers (Figure 1). Forty-four of the patients in the cohort had RNA-based Nanostring immuno-oncology assay data available. We were able to obtain paired quantitative IHC and Nanostring based PD-L1 (CD274) gene expression values for 40 patients and Spearman's correlation was performed (Figure 3). Results: The primary tumor site was tonsil in 29 (56%) patients, 22 (42%) base of tongue, and 1 (2%) soft palate. Females made up 10% of the cohort. There were 9 (17%) current smokers, 20 (38%) former, and 23 (44%) never smokers. Pack-years was available for 22 (76%) of the current and former smokers (median: 30, range: 1-75 pack years). There were 5 patient deaths, and 10 patients experienced recurrence. PD-L1 expression on IHC was not predictive of death or recurrence (p=0.39 and p=0.07, respectively). Median follow up time was 20.5 months (range: 2.7 to 83 months). There was a significant difference in PD-L1 expression on IHC between current smokers and the former/never group (Figure 1). Relative gene expression of CD274 measured by the Nanostring assay was lower in current smokers as compared to former/never smokers, however this trend did not meet significance. There was a strong positive linear correlation between Nanostring based CD274 gene expression and PD-L1 expression on IHC (r=0.68, p &lt 0.0001). Conclusion: Current smoking status at the time of diagnosis is associated with decreased PD-L1 expression as compared to former and never smokers. These results may have implications when considering immune checkpoint inhibition therapy in smokers with HPV(+) OPSCC. Citation Format: Ricardo J. Ramirez, Benjamin Wahle, Zixing Liu, Angela Mazul, Paul Zolkind, Mena Mansour, Krzysztof Hyrc, Jose P. Zevallos. Differences in PD-L1 expression by smoking status in HPV positive oropharyngeal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5241.

Heat shock factor 1 (HSF1), a master regulator of heat shock responses, plays an important role in tumorigenesis. In this study we demonstrated that HSF1 is required for chemotherapeutic agent-induced cytoprotective autophagy through transcriptional up-regulation of autophagy-related gene ATG7. Interestingly, this is independent of the HSF1 heat shock response function. Treatment of cancer cells with the FDA-approved chemotherapeutic agent carboplatin induced autophagy and growth inhibition, which were significantly increased upon knockdown of HSF1. Mechanistic studies revealed that HSF1 regulates autophagy by directly binding to ATG7 promoter and transcriptionally up-regulating its expression. Significantly, breast cancer patient sample study revealed that a higher ATG7 expression level is associated with poor patient survival. This novel finding was further confirmed by analysis of two independent patient databases, demonstrating a prognostic value of ATG7. Furthermore, a strong positive correlation was observed between levels of HSF1 and ATG7 in triple-negative breast cancer patient samples, thus validating our in vitro findings. This is the first study identifying a critical role for HSF1 in controlling cytoprotective autophagy through regulation of ATG7, which is distinct from the HSF1 function in the heat shock response. This is also the first study demonstrating a prognostic value of ATG7 in breast cancer patients. These findings strongly argue that combining chemotherapeutic agents with autophagy inhibition by repressing HSF1/ATG7 axis represents a promising strategy for future cancer treatment.

In many types of cancer, the expression of the immunoregulatory protein B7-H3 has been associated with poor prognosis. Previously, we observed a link between B7-H3 and tumor cell migration and invasion, and in present study, we have investigated the role of B7-H3 in chemoresistance in breast cancer. We observed that silencing of B7-H3, via stable short hairpin RNA or transient short interfering RNA transfection, increased the sensitivity of multiple human breast cancer cell lines to paclitaxel as a result of enhanced drug-induced apoptosis. Overexpression of B7-H3 made the cancer cells more resistant to the drug. Next, we investigated the mechanisms behind B7-H3–mediated paclitaxel resistance and found that the level of Stat3 Tyr705 phosphorylation was decreased in B7-H3 knockdown cells along with the expression of its direct downstream targets Mcl-1 and survivin. The phosphorylation of Janus kinase 2 (Jak2), an upstream molecule of Stat3, was also significantly decreased. In contrast, reexpression of B7-H3 in B7-H3 knockdown and low B7-H3 expressing cells increased the phosphorylation of Jak2 and Stat3. In vivo animal experiments showed that B7-H3 knockdown tumors displayed a slower growth rate than the control xenografts. Importantly, paclitaxel treatment showed a strong antitumor activity in the mice with B7-H3 knockdown tumors, but only a marginal effect in the control group. Taken together, our data show that in breast cancer cells, B7-H3 induces paclitaxel resistance, at least partially by interfering with Jak2/Stat3 pathway. These results provide novel insight into the function of B7-H3 and encourage the design and testing of approaches targeting this protein and its partners. Mol Cancer Ther; 10(6); 960–71. ©2011 AACR.

Paclitaxel (Taxol) is an effective chemotherapeutic agent for treatment of cancer patients. Despite impressive initial clinical responses, the majority of patients eventually develop some degree of resistance to Taxol-based therapy. The mechanisms underlying cancer cells resistance to Taxol are not fully understood. MicroRNA (miRNA) has emerged to play important roles in tumorigenesis and drug resistance. However, the interaction between the development of Taxol resistance and miRNA has not been previously explored. In this study we utilized a miRNA array to compare the differentially expressed miRNAs in Taxol-resistant and their Taxol-sensitive parental cells. We verified that miR-125b, miR-221, miR-222, and miR-923 were up-regulated in Taxol-resistant cancer cells by real-time PCR. We further investigated the role and mechanisms of miR-125b in Taxol resistance. We found that miR-125b was up-regulated in Taxol-resistant cells, causing a marked inhibition of Taxol-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to Taxol in cancer cells. Moreover, we demonstrated that the pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) is a direct target of miR-125b. Down-regulation of Bak1 suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol. Restoring Bak1 expression by either miR-125b inhibitor or re-expression of Bak1 in miR-125b-overexpressing cells recovered Taxol sensitivity, overcoming miR-125-mediated Taxol resistance. Taken together, our data strongly support a central role for miR-125b in conferring Taxol resistance through the suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming Taxol resistance in a number of different tumor histologies.

Gene silencing resulting from aberrant DNA methylation can lead to tumorigenesis. Therefore, drugs that inhibit or interfere with DNA methylation have been used to reactivate and induce silenced gene re-expression in malignancies. Two demethylating agents, azacitidine and decitabine, are approved for the treatment of myelodysplastic syndromes (MDS) by the U.S. Food and Drug Administration (FDA), and are now considered the standard of care in MDS. In this review, we discuss clinical data, including clinical benefits and toxicities, which led to the approval of azacitidine and decitabine. We also summarize findings from clinical trials that used these two demethylating agents in the treatment of solid tumors. Lastly, we discuss some limitations in the use of azacitidine and decitabine in cancer therapy.

B7-H3 is a member of B7 family of immunoregulatory transmembrane glycoproteins expressed by T cells. While B7-H3 overexpression is associated with poor outcomes in multiple cancers, it also has immune-independent roles outside T cells and its precise mechanistic contributions to cancer are unclear. In this study, we investigated the role of B7-H3 in metabolic reprogramming of cancer cells in vitro and in vivo. We found that B7-H3 promoted the Warburg effect, evidenced by increased glucose uptake and lactate production in B7-H3–expressing cells. B7-H3 also increased the protein levels of HIF1α and its downstream targets, LDHA and PDK1, key enzymes in the glycolytic pathway. Furthermore, B7-H3 promoted reactive oxygen species–dependent stabilization of HIF1α by suppressing the activity of the stress-activated transcription factor Nrf2 and its target genes, including the antioxidants SOD1, SOD2, and PRX3. Metabolic imaging of human breast cancer xenografts in mice confirmed that B7-H3 enhanced tumor glucose uptake and tumor growth. Together, our results illuminate the critical immune-independent contributions of B7-H3 to cancer metabolism, presenting a radically new perspective on B7 family immunoregulatory proteins in malignant progression. Cancer Res; 76(8); 2231–42. ©2016 AACR.

Trastuzumab shows remarkable efficacy in treatment of ErbB2-positive breast cancers when used alone or in combination with other chemotherapeutics. However, acquired resistance develops in most treated patients, necessitating alternate treatment strategies. Increased aerobic glycolysis is a hallmark of cancer and inhibition of glycolysis may offer a promising strategy to preferentially kill cancer cells. In this study, we investigated the antitumor effects of trastuzumab in combination with glycolysis inhibitors in ErbB2-positive breast cancer. We found that trastuzumab inhibits glycolysis via downregulation of heat shock factor 1 (HSF1) and lactate dehydrogenase A (LDH-A) in ErbB2-positive cancer cells, resulting in tumor growth inhibition. Moreover, increased glycolysis via HSF1 and LDH-A contributes to trastuzumab resistance. Importantly, we found that combining trastuzumab with glycolysis inhibition synergistically inhibited trastuzumab-sensitive and -resistant breast cancers in vitro and in vivo, due to more efficient inhibition of glycolysis. Taken together, our findings show how glycolysis inhibition can dramatically enhance the therapeutic efficacy of trastuzumab in ErbB2-positive breast cancers, potentially useful as a strategy to overcome trastuzumab resistance. Cancer Res; 71(13); 4585–97. ©2011 AACR.

BackgroundTaxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells.ResultsTaxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells.ConclusionLDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.

It is well accepted that ErbB2, an important oncoprotein, localizes on the plasma membrane as a receptor tyrosine kinase. This study describes a novel observation that ErbB2 localizes in mitochondria of multiple cancer cell lines and patient samples. We identified an endogenous mitochondrial targeting sequence in ErbB2 and found that ErbB2 translocates into mitochondria through the association with mtHSP70, a key player in the canonical mitochondrial protein importation pathway. Additionally, we observed that mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complex I, II and IV of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and the cellular ATP level also were decreased by mtErbB2. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Mitochondrial ErbB2 regulates the phosphorylation and activity of COX II and consequently the cytochrome c release and apoptosis. Additional studies showed that cancer cells with higher levels of mtErbB2 were more resistant to Trastuzumab and deletion of the mitochondrial targeting sequence of ErbB2 sensitized the cells to Trastuzumab. Our study provides a novel perspective on the metabolic regulatory functions of ErbB2 and reveals that mtErbB2 plays an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics. Since ErbB2 plays important roles in multiple organs and multiple types of cancers, the study may have broad impact on the fields of cancer biology. The work was supported by The Vincent F. Kilborn Jr. Cancer Research Foundation and National Institutes of Health Grant RO1CA149646. Citation Format: Yan Ding, Zixing Liu, Shruti Desai, Lewis Pannell, Hong Yi, Elizabath Wright, Laurie Owen, Windy Dean-Colomb, Oystein Fodstad, Jianrong Lu, Ming Tan. Receptor tyrosine kinase ErbB2/HER2 translocates into mitochondria and regulates cellular metabolism. [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 3025. doi:10.1158/1538-7445.AM2013-3025

Breast cancer is the most common type of cancer as well as one of the leading causes of cancer-related deaths in women. The increasing emergence of drug resistance to commonly used chemotherapeutic agents has warranted the development of novel and robust treatment options for cancer patients. Carboplatin, a DNA damaging agent, is one of the therapeutic agents which has shown promising results in breast cancer clinical trials. Carboplatin is a second generation platinum drug, increasingly used in clinic to treat cancer patients because of its reduced toxicity and side effects when compared to Cisplatin. Heat Shock Factor 1 (HSF1) is the master regulator of heat shock responses which has also been shown to regulate certain non-heat shock reponse genes. As a transcription factor HSF1 can function as both an activator and repressor of its target genes, resulting in regulation of critical cellular processes invovled in managing cellular stress. The focus of this study is to identify the role of HSF1 in Carboplatin mediated-sensitivity in breast cancer cells, to provide novel insights to improve treatment strategies for cancer patients. Carboplatin treatment in multiple breast cancer cell lines induced growth inhibition which was significantly enhanced upon HSF1 knockdown. Interestingly, the treatment also induced autophagy in these cells. Autophagy is a type II programmed cell death, which is induced in response to metabolic stress and has been demonstrated to mediate drug resistance, since it can play a protective role and allow cells to survive under stressful conditions. Knockdown of HSF1 along with inhibition of autophagy by 3-Methyladenine (3-MA) led to increase in apoptosis in these cells. Furthermore, HSF1 was required for the induction of autophagy in Carboplatin treated cells. Knockdown of HSF1 by siRNA (transient) and shRNA (stable), inhibited induction of autophagy in the presence of Carboplatin, as measured by the punctate EGFP-LC3 structures as well as acridine orange staining. Chromatin immunoprecipitation (ChIP) assay along with Real time- PCR analysis revealed that HSF1 can regulate autophagy by directly regulating the expression of autophagy related genes in the presence of Carboplatin. These findings indicate that HSF1 activates autophagy in response to the Carboplatin induced cellular stress thus leading to cell survival and reducing sensitivity to Carboplatin. Our data strongly support a critical role for HSF1 in promoting chemoresistance through induction of autophgy. This finding has important implications in the development of targeted therapeutics for overcoming Carboplatin resistance in breast cancer. 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 LB-488. doi:1538-7445.AM2012-LB-488

Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells. Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells. LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2130. doi:10.1158/1538-7445.AM2011-2130

Paclitaxel (Taxol) is one of the most effective chemotherapeutic agents for treatment of cancer patients. Despite impressive initial clinical responses, the majority of patients will eventually develop some degree of resistance to Taxol-based therapy. The mechanisms underlying cancer cells resistance to Taxol are not fully understood. microRNA (miRNA) has emerged to play important roles in tumorigenesis and drug-resistance. However, the interaction between the development of Taxol resistance and miRNA has not been previously explored. In this study, we utilized a miRNA array to screen differentially expressed miRNAs, comparing Taxol-resistant and their Taxol-sensitive parental cells, and investigated the role and mechanisms of miR-125b in Taxol resistance of breast cancer cells. We found that miR-125b was up-regulated in Taxol-resistant cells, with its overexpression causing a marked inhibition of Taxol-induced cytotoxicity and apoptosis, and a subsequent increase in the resistance of cancer cells to Taxol. Moreover, we demonstrated that Bak1 is a direct target of miR-125b in breast cancer cells, and down- regulation of Bak1 suppressed Taxol- induced apoptosis and led to an increased resistance to Taxol. Furthermore, restoring the expression of Bak1 by either miR-125b inhibitor or specific siRNA recovered Taxol sensitivity, overcoming miR-125-mediated Taxol resistance. Taken together, our data strongly supports a central role for miR-125b in conferring Taxol resistance through the suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming Taxol resistance in a number of different tumor histologies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2109.