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HNSCC is the 6th most common malignancy in the world. The prognosis is favorable during early stages; however, the disease is rarely diagnosed early due to the lack of symptoms. Most HNSCC patients present with metastatic disease for which the survival rates remain low. Hypoxia serves as a bad prognostic factor in HNSCC correlating with worse survival and resistance to radiotherapy. We aimed to discover novel targets in metastatic HNSCC utilizing a unique collection of matched sets of cell lines derived from primary tumors and their respective metastatic sites. We carried out expression profiling across the lines to reveal potential common changes in gene expression between the cells derived from primary and metastatic sites in each patient as well as between normoxia and hypoxia. This analysis revealed significant changes in gene expression between the described conditions. Interestingly, beta defensin 2 was one of the genes that came up as overexpressed in metastasis as well as in cells cultured in hypoxia. Beta defensins are small cationic peptides that belong to the innate immune system and exhibit antimicrobial and antiviral activities. Few studies reported their abnormal expression patterns in various cancers including HNSCC. While various novel anticancer therapies are currently being developed, early diagnosis remains one of the biggest challenges in cancer research. A discovery of soluble serum factors that can reliably detect the presence of primary or metastatic disease would serve as an extremely valuable tool in defining diagnosis and prognosis, predicting the response to treatment, and monitoring disease progression. We hypothesized the beta defensin 2 can serve as a serum biomarker of hypoxia and metastasis in HNSCC patients. Utilizing a commercial ELISA kit developed to detect and quantify levels of beta-defensin in human sera, we demonstrated that media collected from cell lines derived from metastases contained higher levels of beta-defensin compared to the cell lines derived from the primary tumors. Moreover, sera from HNSCC patients showed higher levels of beta defensin compared to the normal controls. As a next step, we tested sera samples from 40 HNSCC patients, of whom 20 had lymph node metastasis and 20 did not. While these data are still under analysis, preliminary results suggest that higher concentrations of beta-defensin correlate with the presence of lymph node metastasis in HNSCC patients. Utilizing two large chemical libraries that together contain about 4,000 FDA-approved drugs, we performed high-throughput screening of the HNSCC lines described above in order to discover new drugs targeting head and neck cancer, including drugs that target selectively metastatic cells compared to their primary tumor counterparts. Interestingly, many of the metastasis-specific drugs were antibiotics. Together with the findings described above, these data clearly suggest a connection between patient microbiome and the metastatic process in HNSCC. Citation Format: Maria Kondratyev, Aleksandra Pesic, Carl Virtanen, Marianne Koritzinsky, Brad Wouters. Antibacterial defense and metastatic progression—lessons from head and neck cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B10.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary tumors and their respective metastatic sites, with the goal of identifying genes that become essential for cell survival only following metastasis. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. We are particularly interested in the identification of contextual synthetic lethal oncogenes expressed on the cell surface, as those are easily targetable by therapeutic antibodies. To identify these targets, we performed high-throughput flow cytometry screening that enables evaluation of 370 validated cell surface antibodies. Cell surface targets differentially expressed in metastatic lines included CECAM and CCR6 that were previously reported to be implicated in metastasis and tumor progression as well as Thy1, a known marker of stem cells involved in regulation of cell adhesion. Cell surface targets induced under hypoxic conditions across the cell lines included CA9, an enzyme that is known to regulate pH in hypoxic cells and be associated with tumor progression, as well as CD338, CD264 and CD312, that were previously associated with stemness in a few models of cancer. Interestingly, the described proteins were also found to be differentially essential in the shRNA screens, highlighting their functional importance in tumor progression and hypoxia survival. We are currently investigating the role of these proteins in HNSCC metastasis utilizing our unique collection of matched pairs of HNSCC lines from multiple patients. Moreover, we are utilizing our pipeline of patient derived HNSCC xenografts to test the effect of knocking down the described genes in patient tumors. We are also testing the expression of selected hits in histological sections of patient tumorsthe 400-patient TMA by immunohistochemistry looking for correlation with tumor grade, aggressiveness, levels of hypoxia as well as presence/absence of metastasis in the patient. Citation Format: Maria Kondratyev, Aleksandra Pesic, Azin Sayad, Troy Ketela, Natalie Stickle, Carl Virtanen, Jason Moffat, Laurie Ailles, Marianne Koritzinsky, Brad Wouters. Cell surface targets in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3054.

Head and neck carcinoma (HNSCC) is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. The idea behind this selective targeting is based on discovering molecular pathways that became essential in metastatic cells, and then exploiting this vulnerability through targeted agents. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary tumors and their respective metastatic sites, with the goal of identifying genes that become essential for cell survival only following metastasis. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. To complement the functional screens data, we performed targeted sequencing of the most commonly altered genes in HNSCC as reported by the TCGA profiled gene expression in the HNSCC cell lines cultured under normoxia and hypoxia using Illumina microarrays. These analyses led to the discovery of genes that are differentially essential in metastatic cells as well as in cells exposed to hypoxic conditions. Utilizing CRISPR technology, we assembled a library of guide RNAs targeting the discovered hits and are currently validating the top identified genes using both in vitro and in vivo systems. To validate hits that are essential in metastatic cells in vivo, we engineered selected “matched” pairs of HNSCC cell lines to express CAS9 protein upon induction with doxycycline. The cells are then transduced with the library of guides and injected subcutaneously into mice; tumors from control and doxycycline treated animals are compared. The difference in genes that are essential for growth of tumors seeded by primary tumor derived and metastasis derived HNSCC cell lines is then assessed in a quantitative manner. Citation Format: Maria Kondratyev, Aleksandra Pesic, Troy Ketela, Azin Sayad, Stephano Marastoni, Jason Mofat, Carl Virtanen, Natalie Stickle, Reider Grenman, Brad Wouters. Novel targets in metastatic HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 407.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies, we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Utilizing CRISPR methodology, we established that “fixing” the mutation results in reversal of metastatic phenotype of the cells, making them Notch independent similar to their primary tumor counterparts. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Overall, our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies. Citation Format: Maria Kondratyev, Aleksandra Pesic, Troy Ketela, Azin Sayad, Stephano Marastoni, Carl Virtanen, Laurie Ailles, Soroush Samadian, Mikhail Bashkurov, Marianne Koritzinsky, Brad Wouters. Novel therapeutic targets in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3020. doi:10.1158/1538-7445.AM2017-3020

Aberrant activation of the Notch signaling system and over-expression of the Notch ligand JAG1 are associated with poor outcome in breast cancer. The plasminogen activator system, which includes urokinase-type plasminogen activator (uPA), has been validated as a marker of recurrence, high metastasis risk and death in breast malignancy. By using microarray profiling of breast cancer cell lines that had undergone siRNA-mediated abrogation of Notch signaling we uncovered a link between activated Notch and uPA expression. An association between elevated expression of the Notch ligand JAG1, uPA and the basal breast cancer subtype was confirmed in breast cancer cell lines. The association between JAG1 and uPA expression persisted in a survey of primary carcinomas of the breast. We found that Notch knockdown reduced transcription of uPA and phenocopied uPA knockdown in breast cancer cells. Through mutational analysis we identified a CBF-1 binding site in the uPA promoter that is required for direct transcriptional regulation by Notch. These data suggest that JAG1-induced Notch activation results in breast cancer progression through up-regulation of the plasminogen activator system and directly links these two poor prognosis pathways. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-12.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that metastatic, but not primary tumor cells, undergo apoptosis upon knockdown of Notch3 and that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies. Citation Format: Maria Kondratyev, Aleksandra Pesic, Stephano Marastoni, Troy Ketela, Jason Moffat, Carl Virtanen, Azin Sayad, Mikhail Bashkurov, Alessandro Dati, Laurie Ailles, Reidar Grenman, Marianne Koritzinsky, Brad Wouters. Novel therapeutic targets in head and neck cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3794.

Aberrant activation of the Notch receptor signaling pathway and overexpression of the Notch ligand JAG1 are associated with poor outcome in breast cancer. The plasminogen activator system, which includes urokinase-type plasminogen activator (uPA), has been validated as a marker of recurrence, high metastasis risk and death in breast malignancy. By using microarray profiling of breast cancer cell lines that had undergone siRNA-mediated abrogation of Notch signaling we uncovered a link between activated Notch signaling and uPA expression. An association between elevated expression of the Notch ligand JAG1, uPA, and the basal-like breast cancer subtype was confirmed in breast cancer cell lines. The association between JAG1 and uPA expression persisted in a survey of primary carcinomas of the breast. We found that Notch knockdown reduced transcription of uPA and phenocopied uPA knockdown in breast cancer cells. Through mutational analysis we identified a CBF-1 binding site in the uPA promoter that is required for direct transcriptional regulation by Notch. These data suggest that JAG1-induced Notch activation results in breast cancer progression through upregulation of the plasminogen activator system, directly linking these 2 important pathways of poor prognosis. Cancer Res; 71(1); 277–86. ©2010 AACR.

Introduction: The molecular characterization of patients who exhibit a unique response to treatments that are not effective in most patients, so called “exceptional responders”, can shed light on the biological underpinnings of the response and provide rationale for selection of future patients for the same treatment. Here we present the genomic characterization of a 22-year-old female patient who presented with an unclassifiable kidney cancer and diffuse bilateral lung metastases. The primary tumour was resected and platinum plus gemcitabine therapy achieved a complete resolution of the lung metastases. Approximately 2 years later the patient again developed bilateral lung metastases and an additional course of platinum plus gemcitabine once again achieved a complete radiological response. To better understand the genomic contributors to this dramatic and recurrent response, we performed whole exome sequence analysis of the diagnostic formalin-fixed paraffin-embedded tumor tissue and a matched blood sample. Methods and Results: The patient provided informed consent and was profiled through the Princess Margaret Cancer Centre Genomic Investigation of Unusual and Spectacular responders (GENIUS) program. From tumour and matched blood specimens, we isolated coding and untranslated regions followed by deep sequencing (∼250X median coverage) on an Illumina HiSeq 2000. Copy-number analysis using VarScan2 revealed 23 somatic copy-number alterations (12 gains, 11 losses) including loss of 9p and gains of 3q and 8q, consistent with reports on clear cell renal cell carcinomas. Mutational analysis using muTect found 60 somatic, coding mutations, consistent with mutation rates seen in other kidney cancers. Of these variants, only one was a previously reported hotspot mutation, GNAS p.R201H (NM_000516, also known as p.R844H on transcript NM_080425). This mutation causes constitutive activation of the G-protein complex and activates adenylate cyclase to produce cyclic-AMP (cAMP) that can activate oncogenic pathways. However, excess cellular cAMP levels have also been found to promote apoptosis. Furthermore, a germline SNP in GNAS (rs7121), thought to increase GNAS transcript stability, is associated with improved response to gemcitabine plus platinum in non-small-cell lung cancer patients. The current patient was found to be heterozygous for the rs7121 SNP, which may compound the chemosensitivity phenotype observed. Conclusion: We have identified a p.R201H mutation in GNAS that may both drive tumour progression and confer exceptional chemo-sensitivity in a patient with an unclassified kidney cancer. Further analysis is underway in vitro to validate the hypothesis that this mutation confers sensitivity to chemotherapy. Confirmation of this hypothesis may suggest that chemotherapy could be considered for patients harbouring this mutation. Citation Format: Jeff P. Bruce, Arnavaz Danesh, Neil Winegarden, Patrick Yau, Carl Virtanen, Suzanne Kamel-Reid, Michael H. Roehrl, Anthony M. Joshua, Jennifer Knox, Trevor J. Pugh. Exome analysis of an exceptional responder uncovers an activating GNAS mutation that may confer sensitivity to cytotoxic chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3898. doi:10.1158/1538-7445.AM2015-3898

Background: High-grade serous ovarian cancer is rarely diagnosed at an early and potentially curable stage, effective early detection and preventative strategies are few. Recently the discovery of occult invasive and intraepithelial tubal carcinomas in BRCA1 mutation carriers, who are at high risk of serous cancer, undergoing prophylactic surgery has focused attention on the fallopian tube epithelium as the cell of origin and has led to the reporting of putative serous cancer precursor lesions. However, little is known of the early molecular events of serous oncogenesis, or why cancers in BRCA 1 mutation carriers are found preferentially in tissues which are responsive to reproductive hormones. We hypothesize that molecular alterations are present in morphologically normal tubal epithelium from BRCA1 heterozygotes, and that these changes may reflect the earliest alterations in serous carcinogenesis and may be markers of increased cancer risk as well as targets for risk reduction. To identify cancer predisposition candidate genes of high-risk fallopian tube epithelium, we compared gene expression profiles of microdissected tubal epithelium from BRCA1 mutation carriers, control women and patients with HGSC. Methodology: Snap-frozen tissues were selected from the UHN Biobank; control and BRCA cases controlled for age, ovarian cycle status at surgery, and hormone therapy. Cases included 12 BRCA1 mutation carriers and 12 control women, undergoing salpingo-oophorectomy for reasons other than adnexal malignancy or family history, 15 HGSC and 8 contralateral normal tubes. Epithelium was microdissected in all cases using laser capture, RNA extracted and cDNA amplified. The gene expression profiles were generated using Affymetrix Human Genome U133 Plus 2.0 Array. Candidate genes were validated by qPCR and IHC on 2 fallopian tube TMAs. IHC was scored using Spectrum. Statistical analysis was performed using ANOVA (p Results: We focused the microarray analyses on identifying differential expression between BRCA1 heterozygotes and controls. Comparisons between FTE-nonBRCA and FTE-BRCA, resulted in 440 probe sets with more than 2-FC in gene expression. We selected 5 genes to validate by qPCR and IHC, based on gene ontology and known associations to cancer pathways. Conclusions: These results show that histological normal fallopian tube epithelium from BRCA1 heterozygotes have altered gene expression and these differences are most pronounced in the post-ovulatory phase of the ovarian cycle in pre-menopausal women, suggesting that factors associated with the luteal phase are implicated in the increased risk of HGSC in BRCA1 mutation carriers. We also show that genes involved in inflammation pathways are up-regulated in mutation carriers and that these genetic signatures are maintained in HGSC. 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 2835. doi:10.1158/1538-7445.AM2011-2835

Serous ovarian cancer (SOC) typically presents with advanced disease. Current therapy significantly increases survival, yet nearly all patients recur within five years and die of their disease. The cancer-initiating cell (CIC) hypothesis holds that only a subset of cells have the potential to extensively self-renew and give rise to other tumor cells. As their properties may differ from bulk tumor cells, CIC may be spared by available therapies. Identification and characterization of CIC may lead to more effective therapeutic strategies. Previous reports suggested the presence of ovarian CIC, but these findings require validation in primary human samples. Primary SOC were dissociated and depleted of CD45+ cells. Cell surface (CD133/CD44/CD117/CDCP1/MUC-1/VEGFR2) and functional (ALDH1) markers were examined by flow cytometry (n=105). All markers demonstrated intra-/inter-tumor heterogeneity; however only CD133, VEGFR2 and ALDH marked minority populations in all samples. In contrast to a report that CD44+/CD117+ cells identify ovarian CIC, CD117 was present on only half of SOC samples examined (N=75) and only one quarter had a CD44+/CD117+ population. Limiting dilution analysis of primary SOC injected in the mammary fat pad of NOD/SCID mice (95% take at 106 cells) revealed the CIC frequency in primary tumors (n=13) and metastases (n=6 +5 matched) to be ∼1/40000 (n=13). The CIC frequency was significantly higher (∼1/9000) in primary and recurrent ascites (n=16, p=0.002). Xenografts could be passaged at least 3 times, providing evidence of self-renewal. The CIC frequency remained constant in nearly all xenografts from primary tumors, but increased substantially with passage of recurrences, suggesting greater genetic instability. CD133+ cells from primary tumors (n=2), matched metastases (n=2), ascites (n=6) and passage 1 xenografts (n=6) were enriched for CIC (1/300-1/4000), and all (or the vast majority) of CIC activity resided within the CD133+ fraction. Xenografts from CD133+ cells gave rise to CD133+ and CD133- cells and could be serially passaged at least 1-3x. VEGFR2+ and ALDH1+ cells also were enriched for CIC, to a lower extent than CD133. In contrast, after sorting for CD117/CD44 (n=3), tumors arose from all fractions but CD117+/CD44+ cells. Our data are consistent with a hierarchical model of SOC and indicate that CD133 is a marker for ovarian CIC. Current work is devoted to profiling the CD133+ population and identifying additional markers, using high throughput flow cytometry and a panel of 234 antigens and other methods. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4303.