Hui Sun Leong

Mutational landscapes of tongue carcinoma reveal recurrent mutations in genes of therapeutic and prognostic relevance

BackgroundCarcinoma of the oral tongue (OTSCC) is the most common malignancy of the oral cavity, characterized by frequent recurrence and poor survival. The last three decades has witnessed a change in the OTSCC epidemiological profile, with increasing incidence in younger patients, females and never-smokers. Here, we sought to characterize the OTSCC genomic landscape and to determine factors that may delineate the genetic basis of this disease, inform prognosis and identify targets for therapeutic intervention.MethodsSeventy-eight cases were subjected to whole-exome (n = 18) and targeted deep sequencing (n = 60).ResultsWhile the most common mutation was in TP53, the OTSCC genetic landscape differed from previously described cohorts of patients with head and neck tumors: OTSCCs demonstrated frequent mutations in DST and RNF213, while alterations in CDKN2A and NOTCH1 were significantly less frequent. Despite a lack of previously reported NOTCH1 mutations, integrated analysis showed enrichments of alterations affecting Notch signaling in OTSCC. Importantly, these Notch pathway alterations were prognostic on multivariate analyses. A high proportion of OTSCCs also presented with alterations in drug targetable and chromatin remodeling genes. Patients harboring mutations in actionable pathways were more likely to succumb from recurrent disease compared with those who did not, suggesting that the former should be considered for treatment with targeted compounds in future trials.ConclusionsOur study defines the Asian OTSCC mutational landscape, highlighting the key role of Notch signaling in oral tongue tumorigenesis. We also observed somatic mutations in multiple therapeutically relevant genes, which may represent candidate drug targets in this highly lethal tumor type.

An eleven gene molecular signature for extra-capsular spread in oral squamous cell carcinoma serves as a prognosticator of outcome in patients without nodal metastases.

OBJECTIVES Extracapsular spread (ECS) is an important prognostic factor for oral squamous cell carcinoma (OSCC) and is used to guide management. In this study, we aimed to identify an expression profile signature for ECS in node-positive OSCC using data derived from two different sources: a cohort of OSCC patients from our institution (National Cancer Centre Singapore) and The Cancer Genome Atlas (TCGA) head and neck squamous cell carcinoma (HNSCC) cohort. We also sought to determine if this signature could serve as a prognostic factor in node negative cancers. MATERIALS AND METHODS Patients with a histological diagnosis of OSCC were identified from an institutional database and fresh tumor samples were retrieved. RNA was extracted and gene expression profiling was performed using the Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray platform. RNA sequence data and corresponding clinical data for the TCGA HNSCC cohort were downloaded from the TCGA Data Portal. All data analyses were conducted using R package and SPSS. RESULTS We identified an 11 gene signature (GGH, MTFR1, CDKN3, PSRC1, SMIM3, CA9, IRX4, CPA3, ZSCAN16, CBX7 and ZFP3) which was robust in segregating tumors by ECS status. In node negative patients, patients harboring this ECS signature had a significantly worse overall survival (p=0.04). CONCLUSIONS An eleven gene signature for ECS was derived. Our results also suggest that this signature is prognostic in a separate subset of patients with no nodal metastasis Further validation of this signature on other datasets and immunohistochemical studies are required to establish utility of this signature in stratifying early stage OSCC patients.

Paracrine Factors of Human Fetal MSCs Inhibit Liver Cancer Growth Through Reduced Activation of IGF-1R/PI3K/Akt Signaling

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death in the world. The multikinase inhibitor sorafenib only demonstrated marginal improvement in overall survival for advanced disease prompted the search for alternative treatment options. Human mesenchymal stem cells (MSCs) have the ability to home to tumor cells. However, its functional roles on the tumor microenvironment remain controversial. Herein, we showed that conditioned media derived from human fetal MSC (CM-hfMSCs) expressed high level of the insulin growth factor binding proteins IGFBPs and can sequester free insulin-like growth factors (IGFs) to inhibit HCC cell proliferation. The inhibitory effect of IGFBPs on IGF signaling was further evident from the reduction of activated IGF-1R and PI3K/Akt, leading eventually to the induction of cell cycle arrest. We also demonstrated that CM-hfMSCs could enhance the therapeutic efficacy of sorafenib and sunitinib. To the best of our knowledge, this is the first report to show that CM-hfMSCs has a tumor-specific, antiproliferative effect that is not observed with normal human hepatocyte cells and patient-derived matched normal tissues. Our results thus suggest that CM-hfMSCs can provide a useful tool to design alternative/adjuvant treatment strategies for HCC, especially in related function to potentiate the effects of chemotherapeutic drugs.

Targeting Cancer Stem Cell Plasticity Through Modulation of Epidermal Growth Factor and Insulin-Like Growth Factor Receptor Signaling in Head and Neck Squamous Cell Cancer

Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resistance. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the maintenance and repopulation of CSCs. Tumor spheres were established using patient‐derived cell lines via anchorage‐independent cell culture techniques. These tumor spheres were found to have higher aldehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel‐like factor 4, SRY (sex determining region Y)‐box 2, and Nanog and were resistant to γ‐radiation, 5‐fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD fractions. ALDHigh clones showed higher expression of stem cell and epithelial‐mesenchymal transition markers compared with ALDLow clones. ALD fractions, representing stem cell fractions, fluctuated with serial passaging, equilibrating at a level specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin‐like growth factor‐1 receptor (IGF‐1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF‐1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF‐1R pathways and potentially amenable to targeted therapeutics.

GNA13 expression promotes drug resistance and tumor-initiating phenotypes in squamous cell cancers

Treatment failure in solid tumors occurs due to the survival of specific subpopulations of cells that possess tumor-initiating (TIC) phenotypes. Studies have implicated G protein-coupled-receptors (GPCRs) in cancer progression and the acquisition of TIC phenotypes. Many of the implicated GPCRs signal through the G protein GNA13. In this study, we demonstrate that GNA13 is upregulated in many solid tumors and impacts survival and metastases in patients. GNA13 levels modulate drug resistance and TIC-like phenotypes in patient-derived head and neck squamous cell carcinoma (HNSCC) cells in vitro and in vivo. Blockade of GNA13 expression, or of select downstream pathways, using small-molecule inhibitors abrogates GNA13-induced TIC phenotypes, rendering cells vulnerable to standard-of-care cytotoxic therapies. Taken together, these data indicate that GNA13 expression is a potential prognostic biomarker for tumor progression, and that interfering with GNA13-induced signaling provides a novel strategy to block TICs and drug resistance in HNSCCs.

Single-cell RNA-seq unveils divergent modes of chemoresistance in squamous cell carcinoma

Gastrulation was arguably the key evolutionary innovation that enabled metazoan diversification, leading to the formation of distinct germ layers and specialized tissues. Differential gene expression specifying cell fate is governed by the inputs of intracellular and/or extracellular signals. Beta-catenin/Tcf and the TGF-beta Bone Morphogenetic Protein (BMP) provide critical molecular signaling inputs during germ layer specification in bilaterian metazoans but there has beenno direct experimental evidence for a specific role for BMP signaling during endomesoderm specification in the early branching metazoan Nematostella vectensis (ananthozoan cnidarian),. Using forward transcriptomics, we show that Betacatenin/Tcf signaling and BMP2/4 signaling provide differential inputs into the cnidarian endomesodermal gene regulatory network at the onset of gastrulation (24hpf) inNematostella vectensis. Surprisingly, Beta-catenin/Tcf signaling and BMP2/4 signaling regulate a subset of common downstream target genes in the GRN in opposite ways, leading to the spatial and temporal differentiation of fields of cells in the developing embryo. Thus, we show that regulatory interactions between Beta-catenin/Tcf signaling and BMP2/4 signaling are required for the specification and determination of different embryonic regions and the patterning of the oralaboral axis in Nematostella. We also show functionally that the conserved “kernel” of the bilaterian heart mesoderm GRN is operational in Nematostella vectensis, which reinforces the hypothesis that the endoderm and mesoderm in triploblastic bilaterians evolved from the bifunctional endomesoderm (gastrodermis) of a diploblastic ancestor, and that slow rhythmic contractions might have been one of the earliest functions of mesoderm.

Abstract 3887: GNA13 is a theranostic target that drives drug resistance and cancer stem-like phenotypes in solid tumors

Treatment failure in solid tumors occurs due to the survival of specific subpopulations of cells that possess stem cell-like (CSC) phenotypes. Studies have implicated G protein-coupled-receptors (GPCRs) in cancer progression and the acquisition of aggressive phenotypes. Many of the implicated GPCRs signal through the G12 subfamily, comprised of GNA12 and GNA13. In this study, we demonstrate that GNA13 is upregulated in many solid tumors and impacts survival and metastases in these patients. Consistent with this, we show that GNA13 expression modulates drug resistance through its effect on the CSC sub-population in a panel of patient-derived head and neck (HNSCC) and breast cancer cells. These data were validated in vivo, where GNA13 over-expression in patient-derived xenografts increased tumor initiating capacity, tumorigenicity and drug resistance, with no effect on growth or proliferation. Signaling through NFKB and MAPK pathways appear to be critical to the observed phenotype. Importantly, blockade of GNA13 expression, or select downstream pathways using small-molecule inhibitors, abrogates GNA13-induced CSCs, rendering cells vulnerable to standard-of-care cytotoxic therapy for these cancers. Taken together, these data indicate that GNA13 expression is a potential prognostic biomarker, and interfering with GNA13-induced signaling provides a novel strategy to block CSCs and drug resistance in solid tumors. Citation Format: Suhail Ahmed Kabeer Rasheed, Hui Sun Leong, Manikandan Lakshmanan, Anandhkumar Raju, Dhivya Dadlani, Fui-Teen Chong, Ravisankar Rajarethinam, Thakshayeni Skanthakumar, Ern Yu Tan, Jacqueline Siok Gek Hwang, Kok Hing Lim, Daniel Shao-Weng Tan, Paolo Ceppi, Mei Wang, Vinay Tergaonkar, Patrick J. Casey, N. Gopalakrishna Iyer. GNA13 is a theranostic target that drives drug resistance and cancer stem-like phenotypes in solid tumors [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 3887. doi:10.1158/1538-7445.AM2017-3887

Abstract 2505: GNA13 induction of stemness and resistance to chemotherapy in breast and head and neck cancers

Despite advancement in cancer therapy, the ability of cancer cells to evade therapy often leads to tumor recurrence and distant metastasis, and ultimately to the death of the patient. Increasing evidence shows that these properties of recurrence can be attributed to a small population of cells within the tumor termed cancer stem cells (CSCs). Understanding the mechanisms that support the growth of CSCs could provide new tools to target these cells and improve therapeutic outcomes. G12 proteins, comprised of Gα12 (GNA12) and Gα13 (GNA13) are mediators of G protein coupled receptor (GPCR) signaling. These proteins are highly expressed in several cancers, and blocking their signaling can significantly inhibit cancer cell invasion and metastasis. Recently, we have shown that loss of GNA13 alone can suppress invasion of breast and prostate cancer cells. Moreover, we have recently obtained evidence that enhanced expression of GNA13 is capable of inducing a stem cell phenotype in cancer cells. Specifically, enforced expression of GNA13 in cancer cells containing low levels of GNA13 protein significantly induced CSC population as shown by increased aldehyde dehydrogenase (ALDH1) activity, increased CD44+/CD24− cells, and enhancement of tumor sphere formation. In contrast, in cancer cells that express high levels of GNA13, knockdown of GNA13 suppressed ALDH1 activity, the CD44+/CD24− cell population, and the formation of spheres. Using reporter assays we have identified that GNA13 induced CSC phenotype most probably via activating Jun Kinase - AP1 signaling pathway and blocking this pathway could significantly inhibit CSC phenotype induced by GNA13. Most importantly we have identified that GNA13 expressing cells are resistant to most commonly used chemotherapeutic agents and ionizing radiations. Blocking GNA13 using shRNAs sensitized these cells to both chemo- and radiotherapy significantly. We therefore hypothesize that GNA13 plays a pivotal role in the induction of CSCs and drug resistance, and the suppression of GNA13 activity may provide therapeutic advantage to cancer therapy. Citation Format: Hui Sun Leong, Suhail Ahmed Kabber Rasheed, Manikandan Lakshmanan, Anandh Kumar Raju, Vinay Tergaonkar, Daniel shao-Weng Tan, Patrick Casey, N.Gopalakrishna Iyer. GNA13 induction of stemness and resistance to chemotherapy in breast and head and neck cancers. [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 2505.

Abstract 3874: Mutational landscapes of oral tongue squamous cell carcinoma reveal recurrent mutations in genes of therapeutic and prognostic relevance

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Carcinoma of the oral tongue (OTSCC) is the most common malignancy of the oral cavity, characterized by frequent recurrence and poor survival. The last three decades has witnessed a change in the OTSCC epidemiological profile, with increasing incidence in younger patients, females and never-smokers. This study aims to characterize the OTSCC genomic landscape and to determine factors that may delineate the genetic basis of this disease, inform prognosis and identify targets for therapeutic intervention. Seventy-eight Asian OTSCC cases were subjected to whole-exome and targeted deep sequencing. While the most common mutation seen was in TP53, the OTSCC genetic landscape differed from previously described cohorts of patients with head and neck squamous cell cancers: OTSCCs demonstrated frequent mutations in DST, FSIP2 and RNF213 while alterations in CDKN2A and NOTCH1 were significantly less frequent than previously reported. Despite a lack of previously-known NOTCH1 mutations, integrated analysis showed enrichments of alterations affecting Notch signaling, more commonly seen in women. Importantly, Notch pathway alterations were prognostic on uni- and multivariate analyses. Furthermore, a high proportion of OTSCCs presented with alterations in drug targetable genes and chromatin remodeling genes. Mutations in these groups were also prognostic, where patients harboring mutations in these actionable pathways more likely to succumb from recurrent disease compared to those who did not, suggesting that these patients should indeed be considered for treatment with targeted compounds in future trial designs. In conclusion, these findings define the mutational landscape of OTSCC, highlight the key role of the Notch signaling pathway in oral tongue tumorigenesis and uncover somatic mutations in therapeutically relevant genes which may represent candidate drug targets. Citation Format: Andre L. Vettore, Kalpana Ramnarayanan, Choon Kiat Ong, Hui Sun Leong, Weng Khong Lim, Ioana Cutcutache, John R. Mcpherson, Shenli Zhang, Thankshayeni Skanthakumar, Daniel SW Tan, Bin Tean Teh, Steve Rozen, Patrick Tan, N Gopalakrishna Iyer. Mutational landscapes of oral tongue squamous cell carcinoma reveal recurrent mutations in genes of therapeutic and prognostic relevance. [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 3874. doi:10.1158/1538-7445.AM2015-3874

Abstract 258: Growth factor signaling is critical in the maintenance of cancer stem cell population in head and neck squamous cell cancer.

Tumor heterogeneity is an important concept in the carcinogenesis of solid tumors, and forms the background for the cancer-stem cell (CSCs) hypothesis. Emerging data suggests however that there is plasticity within various cancer sub-populations, and that CSCs exists in a contextually regulated equilibrium, which is multi-directional and less rigid than previously hypothesized. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCC), and identify factors that play a role in the maintenance and re-population of CSCs. Tumor spheres were established using patient-derived tumor cells via anchorage-independent cell culture techniques. RT-PCR showed higher expression of KLF4, SOX2 and Nanog in tumor spheres compared to monolayer culture cells. Cells grown as tumor spheres were more resistant to γ-radiation, 5-fluorouracil, cisplatin and etoposide treatment compared to cells grown in monolayer culture. Using the ALDEFLUOR assays to identify CSCs, we showed that more cells in tumor spheres had high aldehyde dehydrogenase (ALD+ cells) activity. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD+ fractions. As expected, ALDHigh clones showed higher expression of stem cell markers Oct4, Bmi1 and Nanog and EMT markers N-Cadherin, Slug, Snail, β-Catenin, Vimentin compared to ALDLow clones. When ALDHigh clones from HNSCC cells were cultured over time, ALD+ fractions were found to reduce gradually and equilibrate at a specified ALD+ fraction specific to each cell line grown in regular culture media. When growth factors EGF and/or insulin were added to the media, ALD+ fractions could be maintained at higher levels as compared to baseline. ALDHigh clones also showed increased EGFR and IGF-1R phosphorylation when grown in regular culture conditions, with increased activation of downstream factors involving the AKT and ERK pathways compared with ALDLow clones. Blocking these pathways using specific inhibitors against EGFR and IGFR reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, where the maintenance of the stem cell fraction was dependent on the EGF and IGF pathways, which are therefore vulnerable to therapeutic targeting. Citation Format: Hui Sun Leong, Pui Hoon Sew, Fui Teen Chong, Daniel Shao-Weng Tan, Narayanan Gopalakrishna Iyer. Growth factor signaling is critical in the maintenance of cancer stem cell population in head and neck squamous cell cancer. [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 258. doi:10.1158/1538-7445.AM2013-258