HER3 targeting potentiates growth suppressive effects of the PI3K inhibitor BYL719 in pre-clinical models of head and neck squamous cell carcinoma.

BYL719 is a PI3K inhibitor that has demonstrated efficacy in the treatment of head and neck squamous cell carcinoma. BYL719 exerts its therapeutic effect by suppressing AKT and other proliferative signaling mechanisms. Despite PI3K inhibition and AKT suppression, residual activity of protein S6, a core marker of proliferative activation, has been observed. HER3, either via dimerization or activation by its ligand neurgeulin (NRG), is known to activate PI3K. Thus, we hypothesized that co-targeting HER3 and PI3K would lead to greater suppression of the PI3K-AKT signaling pathway and greater tumor suppression than with BYL719 alone. We investigated biochemical expression and activation of the HER3-PI3K-AKT-S6 pathway in HNSCC cell lines and patient-derived xenografts (PDXs). Antitumor effects of HER3 and PI3K inhibitors alone and in combination were evaluated in cell culture and murine models. Treatment of HNSCC cell lines with BYL719 significantly reduced AKT activation and suppressed tumor growth. However, S6 was persistently activated despite suppression of AKT. Combination treatment with KTN3379, a monoclonal antibody targeted against HER3, and BYL719 led to enhanced suppression of in vitro and in vivo cancer growth and durable suppression of AKT and S6. Therefore, inhibition of HER3 with KTN3379 enhanced the effects of PI3K inhibition in pre-clinical HNSCC models. These data support co-targeting HER3 and PI3K for the treatment of HSNCC.

Cross-talk Signaling between HER3 and HPV16 E6 and E7 Mediates Resistance to PI3K Inhibitors in Head and Neck Cancer.

Human papillomavirus (HPV) type 16 is implicated in approximately 75% of head and neck squamous cell carcinomas (HNSCC) that arise in the oropharynx, where viral expression of the E6 and E7 oncoproteins promote cellular transformation, tumor growth, and maintenance. An important oncogenic signaling pathway activated by E6 and E7 is the PI3K pathway, a key driver of carcinogenesis. The PI3K pathway is also activated by mutation or amplification of PIK3CA in over half of HPV(+) HNSCC. In this study, we investigated the efficacy of PI3K-targeted therapies in HPV(+) HNSCC preclinical models and report that HPV(+) cell line- and patient-derived xenografts are resistant to PI3K inhibitors due to feedback signaling emanating from E6 and E7. Receptor tyrosine kinase profiling indicated that PI3K inhibition led to elevated expression of the HER3 receptor, which in turn increased the abundance of E6 and E7 to promote PI3K inhibitor resistance. Targeting HER3 with siRNA or the mAb CDX-3379 reduced E6 and E7 abundance and enhanced the efficacy of PI3K-targeted therapies. Together, these findings suggest that cross-talk between HER3 and HPV oncoproteins promotes resistance to PI3K inhibitors and that cotargeting HER3 and PI3K may be an effective therapeutic strategy in HPV(+) tumors.Significance: These findings suggest a new therapeutic combination that may improve outcomes in HPV(+) head and neck cancer patients. Cancer Res; 78(9); 2383-95. ©2018 AACR.

TMEM16A/ANO1 Inhibits Apoptosis Via Downregulation of Bim Expression.

Purpose: TMEM16A is a calcium-activated chloride channel that is amplified in a variety of cancers, including 30% of head and neck squamous cell carcinomas (HNSCCs), raising the possibility of an anti-apoptotic role in malignant cells. This study investigated this using a multimodal, translational investigation.Experimental Design: Combination of (i) in vitro HNSCC cell culture experiments assessing cell viability, apoptotic activation, and protein expression (ii) in vivo studies assessing similar outcomes, and (iii) molecular and staining analysis of human HNSCC samples.Results: TMEM16A expression was found to correlate with greater tumor size, increased Erk 1/2 activity, less Bim expression, and less apoptotic activity overall in human HNSCC. These findings were corroborated in subsequent in vitro and in vivo studies and expanded to include a cisplatin-resistant phenotype with TMEM16A overexpression. A cohort of 41 patients with laryngeal cancer demonstrated that cases that recurred after chemoradiation failure were associated with a greater TMEM16A overexpression rate than HNSCC that did not recur.Conclusions: Ultimately, this study implicates TMEM16A as a contributor to tumor progression by limiting apoptosis and as a potential biomarker of more aggressive disease. Clin Cancer Res; 23(23); 7324-32. ©2017 AACR.

TMEM16A/ANO1 suppression improves response to antibody-mediated targeted therapy of EGFR and HER2/ERBB2.

TMEM16A, a Ca2+ -activated Cl- channel, contributes to tumor growth in breast cancer and head and neck squamous cell carcinoma (HNSCC). Here, we investigated whether TMEM16A influences the response to EGFR/HER family-targeting biological therapies. Inhibition of TMEM16A Cl- channel activity in breast cancer cells with HER2 amplification induced a loss of viability. Cells resistant to trastuzumab, a monoclonal antibody targeting HER2, showed an increase in TMEM16A expression and heightened sensitivity to Cl- channel inhibition. Treatment of HNSCC cells with cetuximab, a monoclonal antibody targeting EGFR, and simultaneous TMEM16A suppression led to a pronounced loss of viability. Biochemical analyses of cells subjected to TMEM16A inhibitors or expressing chloride-deficient forms of TMEM16A provide further evidence that TMEM16A channel function may play a role in regulating EGFR/HER2 signaling. These data demonstrate that TMEM16A regulates EGFR and HER2 in growth and survival pathways. Furthermore, in the absence of TMEM16A cotargeting, tumor cells may acquire resistance to EGFR/HER inhibitors. Finally, targeting TMEM16A improves response to biological therapies targeting EGFR/HER family members.

TMEM16A/ANO1 is differentially expressed in HPV-negative versus HPV-positive head and neck squamous cell carcinoma through promoter methylation.

Head and neck squamous cell carcinoma (HNSCC) has a variety of causes. Recently, the human papilloma virus (HPV) has been implicated in the rising incidence of oropharyngeal cancer and has led to variety of studies exploring the differences between HPV-positive and HPV-negative HNSCC. The calcium-activated chloride channel TMEM16A is overexpressed in a variety of cancers, including HNSCC, but whether or not it plays different roles in HPV-positive and HPV-negative HNSCC is unknown. Here, we demonstrate that TMEM16A is preferentially overexpressed in HPV-negative HNSCC and that this overexpression of TMEM16A is associated with decreased patient survival. We also show that TMEM16A expression is decreased in HPV-positive HNSCC at the DNA, RNA, and protein levels in patient samples as well as cell lines. We demonstrate that the lower levels of TMEM16A expression in HPV-positive tumors can be attributed to both a combination of copy number alteration and promoter methylation at the DNA level. Additionally, our cellular data show that HPV-negative cell lines are more dependent on TMEM16A for survival than HPV-positive cell lines. Therefore, we suspect that the down-regulation of TMEM16A in HPV-positive HNSCC makes TMEM16A a poor therapeutic target in HPV-positive HNSCC, but a potentially useful target in HPV-negative HNSCC.

ANO1/TMEM16A interacts with EGFR and correlates with sensitivity to EGFR-targeting therapy in head and neck cancer.

The epidermal growth factor receptor (EGFR) contributes to the pathogenesis of head&neck squamous cell carcinoma (HNSCC). However, only a subset of HNSCC patients benefit from anti-EGFR targeted therapy. By performing an unbiased proteomics screen, we found that the calcium-activated chloride channel ANO1 interacts with EGFR and facilitates EGFR-signaling in HNSCC. Using structural mutants of EGFR and ANO1 we identified the trans/juxtamembrane domain of EGFR to be critical for the interaction with ANO1. Our results show that ANO1 and EGFR form a functional complex that jointly regulates HNSCC cell proliferation. Expression of ANO1 affected EGFR stability, while EGFR-signaling elevated ANO1 protein levels, establishing a functional and regulatory link between ANO1 and EGFR. Co-inhibition of EGFR and ANO1 had an additive effect on HNSCC cell proliferation, suggesting that co-targeting of ANO1 and EGFR could enhance the clinical potential of EGFR-targeted therapy in HNSCC and might circumvent the development of resistance to single agent therapy. HNSCC cell lines with amplification and high expression of ANO1 showed enhanced sensitivity to Gefitinib, suggesting ANO1 overexpression as a predictive marker for the response to EGFR-targeting agents in HNSCC therapy. Taken together, our results introduce ANO1 as a promising target and/or biomarker for EGFR-directed therapy in HNSCC.

Choline phosphate cytidylyltransferase-α is a novel antigen detected by the anti-ERCC1 antibody 8F1 with biomarker value in patients with lung and head and neck squamous cell carcinomas.

BACKGROUND: The determination of in situ protein levels of ERCC1 with the 8F1 monoclonal antibody is prognostic of survival in patients with non-small cell lung cancer (NSCLC). The authors previously demonstrated that 8F1 recognizes a second nuclear antigen. This antigen was identified and its value as a biomarker of clinical outcomes analyzed. METHODS: The second antigen was identified by mass spectrometry. Protein identity and antibody specificity were confirmed through knockdown and overexpression experiments. Immunohistochemistry of 187 early-stage NSCLC samples and 60 head and neck squamous cell carcinomas (HNSCCs) was used to examine the influence of the second antigen on 8F1 immunoreactivity and its association with patient outcomes. RESULTS: Choline phosphate cytidylyltransferase-α (CCTα, also known as phosphate cytidylyltransferase 1 choline alpha [PCYT1A], a phospholipid synthesis enzyme regulated by RAS) is the second antigen recognized by 8F1. In NSCLC samples, CCTα contributed (rho, 0.38) to 8F1 immunoreactivity. In samples of squamous cell carcinomas of the lung, CCTα was found to be the dominant determinant of 8F1 immunoreactivity, whereas its contribution in other subtypes of lung cancer was negligible. High expression of CCTα, but not ERCC1, was found to be prognostic of longer disease-free survival (log-rank P = .002) and overall survival (log-rank P = .056). Similarly, in patients with HNSCC, CCTα contributed strongly to 8F1 immunoreactivity (rho, 0.74), and high CCTα expression was found to be prognostic of survival (log-rank P = .022 for disease-free survival and P = .027 for overall survival). CONCLUSIONS: CCTα is the second antigen detected by 8F1. High CCTα expression appears to be prognostic of survival in patients with NSCLC who are treated by surgery alone and patients with HNSCC. CCTα is a promising biomarker of patient survival and deserves further study.

The role of autophagy in alpha-1-antitrypsin deficiency.

In the classical form of alpha-1-antitrypsin (AT) deficiency, a mutant protein accumulates in the endoplasmic reticulum of liver cells, causing hepatic fibrosis and hepatocellular carcinoma by a gain-of-toxic function mechanism. Autophagy is specifically activated by the accumulation of mutant AT, and the autophagy plays a key role in intracellular degradation of this mutant protein. Our recent study indicates that an autophagy enhancer drug can decrease the hepatic load of mutant AT and reduce hepatic fibrosis in a mouse model of AT deficiency. In this chapter, we discuss what is known about autophagy in AT deficiency and methods for characterizing autophagy in cell lines and animal models.

An autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin Z and reduces hepatic fibrosis.

In the classical form of alpha1-antitrypsin (AT) deficiency, a point mutation in AT alters the folding of a liver-derived secretory glycoprotein and renders it aggregation-prone. In addition to decreased serum concentrations of AT, the disorder is characterized by accumulation of the mutant alpha1-antitrypsin Z (ATZ) variant inside cells, causing hepatic fibrosis and/or carcinogenesis by a gain-of-toxic function mechanism. The proteasomal and autophagic pathways are known to mediate degradation of ATZ. Here we show that the autophagy-enhancing drug carbamazepine (CBZ) decreased the hepatic load of ATZ and hepatic fibrosis in a mouse model of AT deficiency-associated liver disease. These results provide a basis for testing CBZ, which has an extensive clinical safety profile, in patients with AT deficiency and also provide a proof of principle for therapeutic use of autophagy enhancers.