Targeting Hypoxia Sensitizes TNBC to Cisplatin and Promotes Inhibition of Both Bulk and Cancer Stem Cells.

Development of targeted therapies for triple-negative breast cancer (TNBC) is an unmet medical need. Cisplatin has demonstrated its promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with hypoxia that, in turn, promotes cancer stem cell (CSC) enrichment and drug resistance. Therapeutic approaches to attenuate this may lead to increased cisplatin efficacy in the clinic for the treatment of TNBC. In this report we analyzed clinical datasets of TNBC and found that TNBC patients possessed higher levels of EGFR and hypoxia gene expression. A similar expression pattern was also observed in cisplatin-resistant ovarian cancer cells. We, thus, developed a new therapeutic approach to inhibit EGFR and hypoxia by combination treatment with metformin and gefitinib that sensitized TNBC cells to cisplatin and led to the inhibition of both CD44+/CD24- and ALDH+ CSCs. We demonstrated a similar inhibition efficacy on organotypic cultures of TNBC patient samples ex vivo. Since these drugs have already been used frequently in the clinic; this study illustrates a novel, clinically translatable therapeutic approach to treat patients with TNBC.

A phase I study of high-dose rosuvastatin with standard dose erlotinib in patients with advanced solid malignancies.

BACKGROUND: Synergistic cytotoxicity with high-dose statins and erlotinib has been demonstrated in preclinical models across a number of tumour types. In this phase I study, we evaluated the safety and potential anti-tumour activity of escalating doses of rosuvastatin in combination with the standard clinical dose of erlotinib in heavily pretreated patients with advanced solid tumours. METHODS: This was a single-center, phase I open-label study to determine the safety and recommended phase two dose (RPTD) of rosuvastatin in combination with 150 mg/day standard dose of erlotinib. Using a 3 + 3 study design and 28-day cycle, escalating doses of rosuvastatin from 1 to 8 mg/kg/day × 2 weeks (cycle 1) and 3 weeks (subsequent cycles) given concurrently with erlotinib were evaluated. In order to expand the experience and to gain additional safety and pharmacokinetic data, two expansions cohorts using concurrent or alternating weekly dosing regimens at the RPTD were also evaluated. RESULTS: All 24 patients enrolled were evaluable for toxicity, and 22 for response. The dose-limiting toxicity (DLT) of reversible muscle toxicity was seen at the 2 mg/kg/day dose level. Maximal tolerated dose (MTD) was determined to be 1 mg/kg/day. Thirty-three percent of patients developed at least 1 ≥ grade 2 muscle toxicity (rhabdomyolysis: 1/24, myalgia: 7/24) resulting in one study-related death. Durable stable disease for more than 170 days was seen in 25 % of patients that received concurrent treatment and were evaluable for response (n = 16). Plasma erlotinib levels on study were unaffected by the addition of rosuvastatin. CONCLUSIONS: The observed disease stabilization rate of 25 % with combination therapy in this heavily pretreated population is encouraging, however, the high levels of muscle toxicities observed limited this combination strategy.

Lovastatin-induced apoptosis is mediated by activating transcription factor 3 and enhanced in combination with salubrinal.

We have previously demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to induce tumor-specific apoptosis. The apoptotic effects of lovastatin were regulated in part by the integrated stress response (ISR) that regulates cellular responses to a wide variety of stress inducers. A key regulator of the ISR apoptotic response is activating transcription factor 3 (ATF3) and its target gene CHOP/GADD153. In our study, we demonstrate that in multiple lovastatin-resistant clones of the squamous cell carcinoma (SCC) cell line SCC9, lovastatin treatment (1-25 µM, 24 hr) in contrast to the parental line failed to significantly induce ATF3 expression. Furthermore, the SCC-derived cell lines SCC25 and HeLa that are sensitive to lovastatin-induced apoptosis also preferentially induce ATF3 expression compared to resistant breast (MCF-7) and prostate carcinoma (PC3)-derived cell lines. In HeLa cells shRNA targeting ATF3 expression as well as in ATF3-deficient murine embryonic fibroblasts, lovastatin-induced cytotoxicity and apoptosis were attenuated. In ex vivo HNSCC tumors, lovastatin also induced ATF3 mRNA expression in two of four tumors evaluated. Salubrinal, an agent that can sustain the activity of a key regulator of the ISR eIF2α, further increased the expression of ATF3 and demonstrated synergistic cytotoxicity in combination with lovastatin in SCC cells. Taken together, our results demonstrate preferential induction of ATF3 in lovastatin-sensitive tumor-derived cell lines that regulate lovastatin-induced apoptosis. Importantly, combining lovastatin with salubrinal enhanced ATF3 expression and induced synergistic cytotoxicity in SCC cells.

Sensitivity of cervical carcinoma cells to vesicular stomatitis virus-induced oncolysis: potential role of human papilloma virus infection.

High-risk carcinogenic subtypes of human papilloma virus (HPV) are associated with the development of squamous cell carcinomas of the cervix (CC) and a subset of head and neck (HNSCC). Recurrent metastatic diseases of these sites display a dismal prognosis. Therefore, there is an urgent need to uncover innovative therapeutic strategies in this clinical setting. Oncolytic viruses, including vesicular stomatitis virus (VSV), were identified due to their ability to specifically target tumor cells that generally display defects in interferon (IFN) signaling. HPV expressed proteins can inhibit IFN signaling; therefore, HPV-infected cells may be particularly sensitive to VSV oncolysis. In this study, we evaluated the sensitivity of four CC (HPV+) and four HNSCC (HPV-) derived cell lines to VSV oncolysis. Interestingly, the CC cell lines were consistently more sensitive to VSV cytotoxicity than the HNSCC cell lines tested. Exogenous IFN addition or infection with two attenuated VSV variants that are more susceptible to IFN inhibition failed to attenuate VSV oncolysis in hypersensitive CC cell lines. Furthermore, the expression of HPV-E6, that inhibits IFN receptor signaling, in the VSV-resistant HNSCC cell line SCC25 attenuated VSV-induced IFN response and significantly enhanced VSV cytotoxicity. Finally, differential VSV infection and replication was confirmed in xenograft murine tumor models and explant tumor tissues from two patients with CC. Taken together, these results demonstrate that HPV-infected cells are susceptible to oncolytic virus therapy and that this approach may represent a novel therapeutic approach in HPV positive CC and HNSCC patients.

The effect of the histone deacetylase inhibitor M344 on BRCA1 expression in breast and ovarian cancer cells.

BACKGROUND: The inhibition of Breast Cancer 1 (BRCA1) expression sensitizes breast and ovarian cancer cells to platinum chemotherapy. However, therapeutically relevant agents that target BRCA1 expression have not been identified. Our recent report suggested the potential of the histone deacetylase (HDAC) inhibitor, M344, to inhibit BRCA1 expression. In this study, we further evaluated the effect of M344 on BRCA1 mRNA and protein expression, as well as its effect on cisplatin-induced cytotoxicity in various breast (MCF7, T-47D and HCC1937) and ovarian (A2780s, A2780cp and OVCAR-4) cancer cell lines. RESULTS: With the addition of M344, the platinum-sensitive breast and ovarian cancer cell lines that displayed relatively high BRCA1 protein levels demonstrated significant potentiation of cisplatin cytotoxicity in association with a reduction of BRCA1 protein. The cisplatin-resistant cell lines, T-47D and A2780s, elicited increased cytotoxicity of cisplatin with M344 and down regulation of BRCA1 protein levels. A2780s cells subjected to combination platinum and M344 treatment, demonstrated increased DNA damage as assessed by the presence of phosphorylated H2A.X foci in comparison to either treatment alone. Using Chromatin Immunoprecipitation, A2780s and MCF7 cells exposed to M344 alone and in combination with cisplatin, did not demonstrate enhanced acetylated Histone 4 at the BRCA1 promoter, suggesting an indirect effect on this promoter. CONCLUSIONS: The enhanced sensitivity of HDAC inhibition to platinum may be mediated through a BRCA1-dependent mechanism in breast and ovarian cancer cells. The findings of this study may be important in the future design of clinical trials involving HDAC inhibitors using BRCA1 as a tumour biomarker.

Activating Transcription Factor 3 regulates in part the enhanced tumour cell cytotoxicity of the histone deacetylase inhibitor M344 and cisplatin in combination.

BACKGROUND: Activating Transcription Factor (ATF) 3 is a key regulator of the cellular integrated stress response whose expression has also been correlated with pro-apoptotic activities in tumour cell models. Combination treatments with chemotherapeutic drugs, such as cisplatin, and histone deacetylase (HDAC) inhibitors have been demonstrated to enhance tumour cell cytotoxicity. We recently demonstrated a role for ATF3 in regulating cisplatin-induced apoptosis and others have shown that HDAC inhibition can also induce cellular stress. In this study, we evaluated the role of ATF3 in regulating the co-operative cytotoxicity of cisplatin in combination with an HDAC inhibitor. RESULTS: The HDAC inhibitor M344 induced ATF3 expression at the protein and mRNA level in a panel of human derived cancer cell lines as determined by Western blot and quantitative RT-PCR analyses. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatments also enhanced the cytotoxic effects of cisplatin in these cancer cell lines. The mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways and dependent on ATF4, a known regulator of ATF3 expression. ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) as well as chromatin immunoprecipitation (ChIP) assays were utilized in determining the mechanistic induction of ATF3 by M344. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. CONCLUSION: This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

Lovastatin induces neuronal differentiation and apoptosis of embryonal carcinoma and neuroblastoma cells: enhanced differentiation and apoptosis in combination with dbcAMP.

Differentiation-based therapeutics are an underutilized but a potentially significant treatment option for cancer patients. We show that lovastatin, a competitive inhibitor of the rate-limiting enzyme of mevalonate synthesis HMG-CoA reductase, is able to induce tumour cell differentiation and apoptosis in vitro. We used embryonal carcinoma (EC) and neuroblastoma (NB) cell lines and found that lovastatin promoted apoptosis and induced expression of the neuronal differentiation markers, tyrosine hydroxylase (TH), and growth-associated protein 43. The apoptotic and differentiation responses were time and dose-dependant and rescued by the co-administration of mevalonate. The expression of TH is regulated primarily by a cyclic AMP (cAMP) response element (CRE) in its promoter. Lovastatin enhanced the expression of a CRE-driven luciferase construct in P19 cells. Furthermore, combining lovastatin with 1 mM dibutyryladenosine 3',5'-cyclic monophosphate treatments induced higher expression from the CRE construct, enhanced differentiation and cytotoxicity. This study suggests the potential of combining these therapeutic approaches in EC and NB patients.

The DNA repair proteins BRCA1 and ERCC1 as predictive markers in sporadic ovarian cancer.

This study compares Breast Cancer 1 (BRCA1) and excision repair cross complementation group 1 (ERCC1) expression as predictive markers and evaluates the in vitro enhancement of platinum sensitivity using targeted agents in sporadic ovarian cancer (OC). A retrospective study was performed of advanced stage OC patients receiving platinum-based chemotherapy. BRCA1 and ERCC1 mRNA expression was determined from frozen tissue of 51 patients. Median overall survival (OS) was longer for patients with lower BRCA1 vs. higher BRCA1 (46 vs.33 months, p = 0.03). High BRCA1 was predictive of poorer OS specifically in patients with residual disease (RD) <2 cm (p = 0.03). There was a non-significant association for patients with lower ERCC1 and RD <2 cm in favor of improved OS and time to progression. Patients who expressed higher levels of both BRCA1 and ERCC1 mRNA had a shorter OS compared to patients with lower levels of either or both transcript (33 vs.46 months, p = 0.04). When Cox proportional modeling was used by representing BRCA1 and ERCC1 mRNA expression as a continuous variable, both emerge as potential predictors of survival. OC cell lines were exposed to chemotherapy in combination with DNA repair pathway inhibitors and cell viability was assessed. In vitro histone deacetylase (HDAC) inhibition increased the sensitivity of A2780s/cp cells to cisplatin and carboplatin but not to taxol, coincident with a significant decrease in BRCA1 and ERCC1 expression, suggesting that this compound directly targets DNA repair. In summary, this study shows that low BRCA1 and ERCC1 expression correlate with improved survival in advanced OC and HDAC inhibition induces synergistic cytotoxicity with platinum in vitro.

C-reactive Protein in HPV-Positive and HPV-Negative Oropharyngeal Cancer.

OBJECTIVE: Evaluate serum C-reactive protein (CRP) in human papillomavirus (HPV)-positive oropharynx cancer as compared with HPV-negative oropharynx cancer and determine if CRP levels were associated with overall survival and/or recurrence-free survival. STUDY DESIGN: Prospective cohort study. SETTING: Tertiary care academic cancer center between 2007 and 2010. SUBJECTS AND METHODS: Among patients with oropharynx cancer and confirmed HPV status, plasma CRP levels were measured with a high-sensitivity ELISA kit. Multivariable logistic regression analysis compared 4 categories of CRP (low, moderate, high, very high) between the HPV-positive and HPV-negative groups. Kaplan-Meier methods and Cox regression models were used to determine overall survival and recurrence-free survival by CRP level in both populations. RESULTS: Between 113 HPV-positive and 110 HPV-negative patients, CRP levels were significantly higher in the HPV-positive group, but these levels did not demonstrate a statistically significant dose-response trend. Higher CRP levels were also associated with reduced overall survival ( P = .016) and recurrence-free survival ( P < .001) within the HPV-negative group in univariable analysis; in multivariate analysis, the comparisons were not significantly different. Within HPV-positive oropharynx cancer, CRP levels were not significantly associated with overall survival or recurrence-free survival in univariable or multivariable analyses. CONCLUSION: Circulating CRP was higher in HPV-positive versus HPV-negative oropharynx cancer. Among HPV-negative patients, higher CRP levels were associated with reduced survival.

Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High-Content Drug Screening.

Cell behavior is highly dependent upon microenvironment. Thus, to identify drugs targeting metastatic cancer, screens need to be performed in tissue mimetic substrates that allow cell invasion and matrix remodeling. A novel biomimetic 3D hydrogel platform that enables quantitative analysis of cell invasion and viability at the individual cell level is developed using automated data acquisition methods with an invasive lung disease (lymphangioleiomyomatosis, LAM) characterized by hyperactive mammalian target of rapamycin complex 1 (mTORC1) signaling as a model. To test the lung-mimetic hydrogel platform, a kinase inhibitor screen is performed using tuberous sclerosis complex 2 (TSC2) hypomorphic cells, identifying Cdk2 inhibition as a putative LAM therapeutic. The 3D hydrogels mimic the native niche, enable multiple modes of invasion, and delineate phenotypic differences between healthy and diseased cells, all of which are critical to effective drug screens of highly invasive diseases including lung cancer.

Activating Transcription Factor 3 as a Novel Regulator of Chemotherapy Response in Breast Cancer.

Anthracyclines, such as doxorubicin, are used as first-line chemotherapeutics, usually in combination therapies, for the treatment of advanced breast cancer. While these drugs have been successful therapeutic options, their use is limited due to serious drug related toxicities and acquired tumor resistance. Uncovering the molecular mechanisms that mediate doxorubicin's cytotoxic effect will lead to the identification of novel more efficacious combination therapies and allow for reduced doses of doxorubicin to be administered while maintaining efficacy. In our study, we demonstrate that activating transcription factor (ATF) 3 expression was upregulated by doxorubicin treatment in a representative panel of human breast cancer cell lines MCF7 and MDA-MB-231. We have also shown that doxorubicin treatment can induce ATF3 expression in ex vivo human breast and ovarian tumor samples. The upregulation of ATF3 in the cell lines was regulated by multiple cellular mechanisms including the activation of JNK and ATM signaling pathways. Importantly, loss of ATF3 expression resulted in reduced sensitivity to doxorubicin treatment in mouse embryonic fibroblasts. Through a 1200 FDA-approved compound library screen, we identified a number of agents whose cytotoxicity is dependent on ATF3 expression that also enhanced doxorubicin induced cytotoxicity. For example, the combination of the HDAC inhibitor vorinostat or the nucleoside analogue trifluridine could synergistically enhance doxorubicin cytotoxicity in the MCF7 cell line. Synergy in cell lines with the combination of ATF3 inducers and patients with elevated basal levels of ATF3 shows enhanced response to chemotherapy. Taken together, our results demonstrate a role for ATF3 in mediating doxorubicin cytotoxicity and provide rationale for the combination of ATF3-inducing agents with doxorubicin as a novel therapeutic approach.

Effect of statin use on oncologic outcomes in head and neck squamous cell carcinoma.

BACKGROUND: Preclinical and early-phase clinical studies have suggested an oncoprotective role of statins in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to determine whether incidental statin use in patients with human papillomavirus (HPV)-negative HNSCC is predictive of improved oncologic outcomes. METHODS: A retrospective cohort study of 1194 patients from the Ontario Cancer Registry diagnosed with HNSCC from 2007 to 2012 was performed using linked databases from the Institute for Clinical Evaluative Sciences. Overall survival (OS) and disease-specific survival (DSS) were compared between patients taking statins and controls. RESULTS: Patients with statin exposure demonstrated improved OS (hazard ratio [HR] 0.758; P = .0011; 95% confidence interval [CI] 0.642-0.896), and DSS (HR 0.693; P = .0040; 95% CI 0.539-0.889) compared with those not on statins at the time of diagnosis. CONCLUSION: Incidental statin use at the time of diagnosis of HPV-negative squamous cell carcinoma (SCC) of the larynx, hypopharynx, and nasopharynx demonstrated improved OS and DSS.

Does metformin usage improve survival in head and neck squamous cell carcinoma? A population-based study.

BACKGROUND: We sought to expand upon preliminary data suggesting that metformin confers a survival benefit to patients with head and neck squamous cell carcinoma (HNSCC). METHODS: A large-scale retrospective cohort study of all patients in Ontario diagnosed with squamous cancer of the larynx, hypopharynx, and nasopharynx between Dec 1st 2007 to Dec 1st 2012 was undertaken. The Institute for Clinical and Evaluative Sciences was accessed to obtain patient demographic, treatment and outcome information. We included patients on metformin at the time of diagnosis. Kaplan Meier methods and Cox Regression models were used. RESULTS: Patients taking metformin at the time of diagnosis had a higher comorbid status but were otherwise similar to patients without metformin usage. Using multivariate analysis, neither overall survival nor disease specific survival was improved in patients on metformin (OS: HR 1.123, p = .338; DSS: HR 1.048, p = .792). CONCLUSIONS: No survival advantage was observed in patients with HNSCC taking metformin at the time of diagnosis.

Correction to: HPV DNA in saliva from patients with SCC of the head and neck is specific for p16-positive oropharyngeal tumours.

Following publication of the original article [1], the authors reported an error in one of the author names. In this Correction the incorrect and correct author names are listed.

Human papillomavirus: An unlikely etiologic factor in sinonasal inverted papilloma.

OBJECTIVES/HYPOTHESIS: Tenuous evidence has supported the hypothesis that sinonasal inverted papilloma (SNIP) arise from human papillomavirus (HPV) infection. To clarify the role of HPV in SNIP, all known HPV sub-types were evaluated by employing a robust polymerase chain reaction-based method in a wide variety of SNIPs from a single institution. STUDY DESIGN: Retrospective surgical specimen tumor sample analysis. METHODS: HPV positivity among SNIP samples and those with squamous cell carcinoma (SCC) were compared. Immunohistochemistry was used to quantify p16 (over)expression among tumors as a surrogate marker for HPV. RESULTS: HPV was detected in 10/76 (13%) SNIP specimens. Identified HPV subtypes included nononcogenic 6 and 11 (6/76, 8%) and oncogenic 16, 18, 45, 56 (4/76, 5%). There was no HPV positivity among SCC samples. Only 4/10 (40%) HPV + samples had > 75% p16 cell staining. CONCLUSION: HPV is not supported as an etiological driver of SNIP development or progression to SCC. The p16 biomarker is not a sensitive indicator of HPV positivity in SNIP. LEVEL OF EVIDENCE: NA Laryngoscope, 2443-2447, 2018.

Dual inhibition of Wnt and Yes-associated protein signaling retards the growth of triple-negative breast cancer in both mesenchymal and epithelial states.

Triple-negative breast cancer (TNBC), the most refractory subtype of breast cancer to current treatments, accounts disproportionately for the majority of breast cancer-related deaths. This is largely due to cancer plasticity and the development of cancer stem cells (CSCs). Recently, distinct yet interconvertible mesenchymal-like and epithelial-like states have been revealed in breast CSCs. Thus, strategies capable of simultaneously inhibiting bulk and CSC populations in both mesenchymal and epithelial states have yet to be developed. Wnt/ß-catenin and Hippo/YAP pathways are crucial in tumorigenesis, but importantly also possess tumor suppressor functions in certain contexts. One possibility is that TNBC cells in epithelial or mesenchymal state may differently affect Wnt/ß-catenin and Hippo/YAP signaling and CSC phenotypes. In this report, we found that YAP signaling and CD44high /CD24-/low CSCs were upregulated while Wnt/ß-catenin signaling and ALDH+ CSCs were downregulated in mesenchymal-like TNBC cells, and vice versa in their epithelial-like counterparts. Dual knockdown of YAP and Wnt/ß-catenin, but neither alone, was required for effective suppression of both CD44high /CD24-/low and ALDH+ CSC populations in mesenchymal and epithelial TNBC cells. These observations were confirmed with cultured tumor fragments prepared from patients with TNBC after treatment with Wnt inhibitor ICG-001 and YAP inhibitor simvastatin. In addition, a clinical database showed that decreased gene expression of Wnt and YAP was positively correlated with decreased ALDH and CD44 expression in patients' samples while increased patient survival. Furthermore, tumor growth of TNBC cells in either epithelial or mesenchymal state was retarded, and both CD44high /CD24-/low and ALDH+ CSC subpopulations were diminished in a human xenograft model after dual administration of ICG-001 and simvastatin. Tumorigenicity was also hampered after secondary transplantation. These data suggest a new therapeutic strategy for TNBC via dual Wnt and YAP inhibition.

Co-inhibition of mTORC1, HDAC and ESR1α retards the growth of triple-negative breast cancer and suppresses cancer stem cells.

Triple-negative breast cancer (TNBC) is the most refractory subtype of breast cancer. It causes the majority of breast cancer-related deaths, which has been largely associated with the plasticity of tumor cells and persistence of cancer stem cells (CSCs). Conventional chemotherapeutics enrich CSCs and lead to drug resistance and disease relapse. Development of a strategy capable of inhibiting both bulk and CSC populations is an unmet medical need. Inhibitors against estrogen receptor 1, HDACs, or mTOR have been studied in the treatment of TNBC; however, the results are inconsistent. In this work, we found that patient TNBC samples expressed high levels of mTORC1 and HDAC genes in comparison to luminal breast cancer samples. Furthermore, co-inhibition of mTORC1 and HDAC with rapamycin and valproic acid, but neither alone, reproducibly promoted ESR1 expression in TNBC cells. In combination with tamoxifen (inhibiting ESR1), both S6RP phosphorylation and rapamycin-induced 4E-BP1 upregulation in TNBC bulk cells was inhibited. We further showed that fractionated CSCs expressed higher levels of mTORC1 and HDAC than non-CSCs. As a result, co-inhibition of mTORC1, HDAC, and ESR1 was capable of reducing both bulk and CSC subpopulations as well as the conversion of fractionated non-CSC to CSCs in TNBC cells. These observations were partially recapitulated with the cultured tumor fragments from TNBC patients. Furthermore, co-administration of rapamycin, valproic acid, and tamoxifen retarded tumor growth and reduced CD44high/+/CD24low/- CSCs in a human TNBC xenograft model and hampered tumorigenesis after secondary transplantation. Since the drugs tested are commonly used in clinic, this study provides a new therapeutic strategy and a strong rationale for clinical evaluation of these combinations for the treatment of patients with TNBC.

Strategies to enhance epidermal growth factor inhibition: targeting the mevalonate pathway.

Mevalonate metabolites play an essential role in transducing epidermal growth factor (EGF) receptor (EGFR)-mediated signaling, as several of these metabolites are required for the function of this receptor and the components of its signaling cascades. Thus, the depletion of mevalonate metabolites may have a significant effect on EGFR function. Lovastatin is a specific and potent inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme of the mevalonate pathway. Targeting 3-hydroxy-3-methylglutaryl CoA reductase using lovastatin induces a potent tumor-specific apoptotic response in a variety of tumor types at therapeutically achievable levels of this drug. The effects of lovastatin on EGFR function and the potential combination effects with EGFR tyrosine kinase inhibitors, such as gefitinib, were evaluated. Lovastatin treatment inhibited EGF-induced EGFR autophosphorylation and its downstream signaling cascades by 24 hours. Combining lovastatin and gefitinib showed enhanced inhibition and cooperative cytotoxicity in a variety of cell lines that included all eight squamous cell carcinomas, four non-small cell lung carcinoma, and four colon carcinoma cell lines tested. Isobologram analyses confirmed that this combination was synergistic, inducing a potent apoptotic response. A phase I study has shown the safety and potential clinical benefit of high-dose lovastatin in patients with recurrent squamous cell carcinoma. The use of lovastatin, which is metabolized by CYP3A4, is contraindicated with drugs, such as gefitinib and erlotinib, which are also metabolized by CYP3A4 due to greatly enhanced toxicity. Rosuvastatin, a relatively novel potent mevalonate pathway inhibitor that is not metabolized significantly by CYP3A4, is a more appropriate statin to combine with either erlotinib or gefitinib. The combination of erlotinib and rosuvastatin has been proposed for a phase I/II study in advanced non-small cell lung carcinoma.

HPV DNA in saliva from patients with SCC of the head and neck is specific for p16-positive oropharyngeal tumours.

BACKGROUND: Human papillomavirus (HPV) is an important cause of head and neck squamous cell carcinoma (HNSCC), especially in young people. These tumours overexpress p16 and respond well to treatment. The rapid detection of HPV in patients with HNSCC may expedite treatment when p16 status is not immediately available. METHODS: Saliva-based DNA collection kits and nested polymerase chain reaction (PCR) were used to determine the HPV status of 62 individuals with biopsy-proven HNSCC. Immunohistochemistry was used to determine tumour p16 status. RESULTS: A total of 62 patients were included in the study. Twenty-nine samples (47%) were positive for HPV DNA, the majority of which were high risk (HR) subtypes (79%). Patients who tested positive for HR HPV were more likely to have a tumour arising in the oropharynx compared to a non-oropharyngeal site (74 vs 26%; p = 0.003). A positive HR HPV saliva assay was 100% specific (95% CI 59-100%) and had a 100% positive predictive value (95% CI 75-100%) for a p16 positive tumour arising in the oropharynx. In contrast, a negative HR HPV assay had a 96% negative predictive value (95% CI 80-100%) for tumours arising in a non-oropharyngeal site. Independent of site, the saliva assay had a sensitivity of 77% (95% CI 54-91%) and a specificity of 94% (95% CI 77-99%), respectively, for a p16 positive tumour. CONCLUSION: We show that a saliva based assay is an effective method for detecting HPV in patients with HNSCC and that a positive HR HPV test is highly specific for p16 positive tumours arising in the oropharynx. This simple and rapid test could be used in cases where a biopsy of the primary tumour is not readily available.

Vulvar Squamous Cell Carcinoma (VSCC) as Two Diseases: HPV Status Identifies Distinct Mutational Profiles Including Oncogenic Fibroblast Growth Factor Receptor 3.

Purpose: Patients with advanced or recurrent invasive vulvar squamous cell carcinoma (VSCC) have limited treatment options and a grave prognosis. Understanding the genomic landscape may facilitate the identification of new therapies and improve clinical outcomes.Experimental Design: A retrospective chart review and molecular analysis of patients with VSCC from 2000 to 2016 was performed at the Ottawa Hospital Research Institute. The presence of oncogenic human papillomavirus (HPV) was determined by nested PCR and amplified DNA was sequenced using the Ion AmpliSeq Cancer Hotspot v2 Panel. The patients were divided into two groups according to HPV status (HPV-positive versus HPV-negative) and clinical outcome correlated with mutation status using descriptive statistics.Results: In 43 VSCC patients, there was a high mutation rate in both HPV-positive (73%) and HPV-negative (90%) disease with the two subgroups expressing distinct genetic profiles. HPV-positive tumors were characterized by oncogenic mutations in PIK3CA (27%), FGFR3 (14%), and PTEN (9%), whereas HPV-negative tumors were found to have mutations in TP53 (57%), HRAS (24%), PI3KCA (19%), and CDKN2A (14%). Mutation S249C in FGFR3 occurred in 14% of HPV-positive tumors. While there were notable differences in the occurrence of TP53, HRAS, PTEN, and FGFR3 mutations according to HPV status, only the rate of TP53 mutations was statistically significant (P = 0.0004). No significant difference in prognosis was found between patients with HPV-positive and HPV-negative VSCC.Conclusions: HPV-positive VSCC is characterized by oncogenic FGFR3 mutations that helps classify this subtype as a separate disease. Inhibitors of FGFR3 merit consideration as a therapeutic strategy in this neglected cancer in women. Clin Cancer Res; 23(15); 4501-10. ©2017 AACR.