Shizhang Ling

An EGFR-ERK-SOX9 Signaling Cascade Links Urothelial Development and Regeneration to Cancer

Like many carcinomas, urothelial carcinoma (UroCa) is associated with chronic injury. A better understanding of this association could inform improved strategies for preventing and treating this disease. We investigated the expression, regulation, and function of the transcriptional regulator SRY-related high-mobility group box 9 (Sox9) in urothelial development, injury repair, and cancer. In mouse bladders, Sox9 levels were high during periods of prenatal urothelial development and diminished with maturation after birth. In adult urothelial cells, Sox9 was quiescent but was rapidly induced by a variety of injuries, including exposure to the carcinogen cyclophosphamide, culture with hydrogen peroxide, and osmotic stress. Activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was required for Sox9 induction in urothelial injury and resulted from activation of the epidermal growth factor receptor (Egfr) by several Egfr ligands that were dramatically induced by injury. In UroCa cell lines, SOX9 expression was constitutively upregulated and could be suppressed by EGFR or ERK1/2 blockade. Gene knockdown showed a role for SOX9 in cell migration and invasion. Accordingly, SOX9 protein levels were preferentially induced in invasive human UroCa tissue samples (n = 84) compared with noninvasive cancers (n = 56) or benign adjacent urothelium (n = 49). These results identify a novel, potentially oncogenic signaling axis linking urothelial injury to UroCa. Inhibiting this axis is feasible through a variety of pharmacologic approaches and may have clinical utility.

Promoter DNA methylation of Oncostatin M receptor-β as a novel diagnostic and therapeutic marker in colon cancer

In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging “cancer methylome”. In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-β (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.

The TGFβ–miR200–MIG6 Pathway Orchestrates the EMT-Associated Kinase Switch That Induces Resistance to EGFR Inhibitors

Although specific mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) identify tumors that are responsive to EGFR tyrosine kinase inhibitors (TKI), these genetic alterations are present in only a minority of patients. Patients with tumors expressing wild-type EGFR lack reliable predictive markers of their clinical response to EGFR TKIs. Although epithelial-mesenchymal transition (EMT) has been inversely correlated with the response of cancers to EGFR-targeted therapy, the precise molecular mechanisms underlying this association have not been defined and no specific EMT-associated biomarker of clinical benefit has been identified. Here, we show that during transforming growth factor β (TGFβ)-mediated EMT, inhibition of the microRNAs 200 (miR200) family results in upregulated expression of the mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR. The MIG6-mediated reduction of EGFR occurs concomitantly with a TGFβ-induced EMT-associated kinase switch of tumor cells to an AKT-activated EGFR-independent state. In a panel of 25 cancer cell lines of different tissue origins, we find that the ratio of the expression levels of MIG6 and miR200c is highly correlated with EMT and resistance to erlotinib. Analyses of primary tumor xenografts of patient-derived lung and pancreatic cancers carrying wild-type EGFR showed that the tumor MIG6(mRNA)/miR200 ratio was inversely correlated with response to erlotinib in vivo. Our data demonstrate that the TGFβ-miR200-MIG6 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors, and identify a low ratio of MIG6 to miR200 as a promising predictive biomarker of the response of tumors to EGFR TKIs.

Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.

MYB rearrangement and clinicopathologic characteristics in head and neck adenoid cystic carcinoma

Salivary gland adenoid cystic carcinoma (ACC) is rare, aggressive, and challenging to treat. Many ACCs have a t(6;9) chromosomal translocation resulting in a MYB‐NFIB fusion gene, but the clinical significance is unclear. The purposes of this study were to describe the clinicopathologic factors impacting survival and to determine the prevalence and clinical significance of MYB‐NFIB fusion.

The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors

The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01).

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.

Epigenetic screening of salivary gland mucoepidermoid carcinoma identifies hypomethylation of CLIC3 as a common alteration

OBJECTIVES The role of promoter methylation in the development of mucoepidermoid carcinoma (MEC) has not been fully explored. In this study, we investigated the epigenetic landscape of MEC. METHODS The Illumina HumanMethylation27 BeadChip array and differential methylation analysis were utilized to screen for epigenetic alterations in 14 primary MEC tumors and 14 matched normal samples. Bisulfite sequencing was used to validate these results, with subsequent quantitative Methylation-Specific PCR (qMSP) to validate chloride intracellular channel protein 3 (CLIC3) in a separate cohort. Furthermore, CLIC3 immunohistochemical (IHC) staining was performed in another separate cohort of MEC. Finally, clinical and pathological characteristics were statistically analyzed for correlation with methylation status of CLIC3 and CLIC3 IHC H-scores by Wilcoxon rank sum, Kruskall-Wallis, and X(2) test tests. RESULTS We obtained 6 significantly differentially methylated gene candidates demonstrating significant promoter hyper- or hypo-methylation from the array data. Using bisulfite sequencing, we found one gene, CLIC3, which showed differential methylation between MEC tumor and normal samples in a small validation cohort. qMSP analysis of the CLIC3 promoter in a separate validation set showed significantly lower methylation level in tumor than in normal. The level of CLIC3 methylation in MECs was not statistically correlated with clinical or pathological characteristics. However, IHC staining intensity and distribution of CLIC3 were significantly increased in MECs, compared with those of normal salivary gland tissues. CONCLUSIONS Hypomethylation of CLIC3 promoter and its overexpression are significant events in MEC. Its functional role and potential therapeutic utility in MEC are worthy of further exploration.

Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

Salivary gland adenoid cystic carcinoma (ACC) is a rare head and neck malignancy without molecular biomarkers that can be used to predict the chemotherapeutic response or prognosis of ACC. The regulation of gene expression of oncogenes and tumor suppressor genes (TSGs) through DNA promoter methylation may play a role in the carcinogenesis of ACC. To identify differentially methylated genes in ACC, a global demethylating agent, 5-aza-2′-deoxycytidine (5-AZA) was utilized to unmask putative TSG silencing in ACC xenograft models in mice. Fresh xenografts were passaged, implanted in triplicate in mice that were treated with 5-AZA daily for 28 days. These xenografts were then evaluated for genome-wide DNA methylation patterns using the Illumina Infinium HumanMethylation27 BeadChip array. Validation of the 32 candidate genes was performed by bisulfite sequencing (BS-seq) in a separate cohort of 6 ACC primary tumors and 6 normal control salivary gland tissues. Hypermethylation was identified in the HCN2 gene promoter in all 6 control tissues, but hypomethylation was found in all 6 ACC tumor tissues. Quantitative validation of HCN2 promoter methylation level in the region detected by BS-seq was performed in a larger cohort of primary tumors (n=32) confirming significant HCN2 hypomethylation in ACCs compared with normal samples (n=10; P=0.04). HCN2 immunohistochemical staining was performed on an ACC tissue microarray. HCN2 staining intensity and H-score, but not percentage of the positively stained cells, were significantly stronger in normal tissues than those of ACC tissues. With our novel screening and sequencing methods, we identified several gene candidates that were methylated. The most significant of these genes, HCN2, was actually hypomethylated in tumors. However, promoter methylation status does not appear to be a major determinant of HCN2 expression in normal and ACC tissues. HCN2 hypomethylation is a biomarker of ACC and may play an important role in the carcinogenesis of ACC.

Abstract 64: Abnormal splicing events in adenoid cystic carcinoma

Salivary gland adenoid cystic carcinoma (ACC) is a relatively rare and understudied malignancy. While this disease can often be controlled locally by surgery and radiation therapy, distant metastases occur in nearly 50% of cases. Interestingly, these metastases can remain asymptomatic for several years, and there are currently no factors that are known to contribute to their dormancy or aggressiveness. Furthermore, there are no targeted chemotherapeutic agents available to treat any stage of this disease. To identify aberrations that contribute to ACC tumorigenesis, we performed whole genome methylation profiling, RNA sequencing, and exome sequencing on 18 primary ACC tumors and matched normal salivary gland tissue. A novel analysis methodology of Splice Expression Variability Analysis (SEVA) coupled with outlier statistical analyses of mRNA splice junctions revealed the presence of several abnormal splicing events (ASEs) in the studied tumors. Interestingly, the ASEs were significantly overrepresented in tumors as compared to matched normal salivary gland tissue. In vitro analyses found that many of the ASEs are also expressed in the only validated ACC cell line, MDA-ACC01, and multiple patient-derived xenograft (PDX) models of ACC. Overall, these studies have identified an unanticipated aberration in ACC and developed a model in which to study ASEs. Future work will be aimed at determining the functional impact that ASEs have on tumor initiation and growth. Citation Format: Brandon Leonard, Joseph O. Humtsoe, Hyun-Su Kim, Shizhang Ling, Luigi Marchionni, Elana J. Fertig, Michael Considine, Patrick Ha. Abnormal splicing events in adenoid cystic carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 64.