Chunxia Cao

Aberrantly activated AREG-EGFR signaling is required for the growth and survival of CRTC1-MAML2 fusion-positive mucoepidermoid carcinoma cells

Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1–MAML2 fusion oncogene, encoded by a recurring chromosomal translocation t(11;19)(q14-21;p12-13), is a frequent genetic alteration found in >50% of mucoepidermoid carcinomas (MEC), the most common malignant SGT. In this study, we aimed to define the role of the CRTC1–MAML2 oncogene in the maintenance of MEC tumor growth and to investigate critical downstream target genes and pathways for therapeutic targeting of MEC. By performing gene expression analyses and functional studies via RNA interference and pharmacological modulation, we determined the importance of the CRTC1–MAML2 fusion gene and its downstream AREG–EGFR signaling in human MEC cancer cell growth and survival in vitro and in vivo using human MEC xenograft models. We found that CRTC1–MAML2 fusion oncogene was required for the growth and survival of fusion-positive human MEC cancer cells in vitro and in vivo. The CRTC1–MAML2 oncoprotein induced the upregulation of the epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) by co-activating the transcription factor CREB, and AREG subsequently activated EGFR signaling in an autocrine manner that promoted MEC cell growth and survival. Importantly, CRTC1–MAML2-positive MEC cells were highly sensitive to EGFR signaling inhibition. Therefore, our study revealed that aberrantly activated AREG–EGFR signaling is required for CRTC1–MAML2-positive MEC cell growth and survival, suggesting that EGFR-targeted therapies will benefit patients with advanced, unresectable CRTC1–MAML2-positive MEC.

cAMP/CREB-regulated LINC00473 marks LKB1- inactivated lung cancer and mediates tumor growth

The LKB1 tumor suppressor gene is frequently mutated and inactivated in non-small cell lung cancer (NSCLC). Loss of LKB1 promotes cancer progression and influences therapeutic responses in preclinical studies; however, specific targeted therapies for lung cancer with LKB1 inactivation are currently unavailable. Here, we have identified a long noncoding RNA (lncRNA) signature that is associated with the loss of LKB1 function. We discovered that LINC00473 is consistently the most highly induced gene in LKB1-inactivated human primary NSCLC samples and derived cell lines. Elevated LINC00473 expression correlated with poor prognosis, and sustained LINC00473 expression was required for the growth and survival of LKB1-inactivated NSCLC cells. Mechanistically, LINC00473 was induced by LKB1 inactivation and subsequent cyclic AMP-responsive element-binding protein (CREB)/CREB-regulated transcription coactivator (CRTC) activation. We determined that LINC00473 is a nuclear lncRNA and interacts with NONO, a component of the cAMP signaling pathway, thereby facilitating CRTC/CREB-mediated transcription. Collectively, our study demonstrates that LINC00473 expression potentially serves as a robust biomarker for tumor LKB1 functional status that can be integrated into clinical trials for patient selection and treatment evaluation, and implicates LINC00473 as a therapeutic target for LKB1-inactivated NSCLC.

Brief Report: Blockade of Notch Signaling in Muscle Stem Cells Causes Muscular Dystrophic Phenotype and Impaired Muscle Regeneration

Muscular dystrophies are a group of devastating diseases characterized by progressive muscle weakness and degeneration, with etiologies including muscle gene mutations and regenerative defects of muscle stem cells. Notch signaling is critical for skeletal myogenesis and has important roles in maintaining the muscle stem cell pool and preventing premature muscle differentiation. To investigate the functional impact of Notch signaling blockade in muscle stem cells, we developed a conditional knock‐in mouse model in which endogenous Notch signaling is specifically blocked in muscle stem cell compartment. Mice with Notch signaling inhibition in muscle stem cells showed several muscular dystrophic features and impaired muscle regeneration. Analyses of satellite cells and isolated primary myoblasts revealed that Notch signaling blockade in muscle stem cells caused reduced activation and proliferation of satellite cells but enhanced differentiation of myoblasts. Our data thus indicate that Notch signaling controls processes that are critical to regeneration in muscular dystrophy, suggesting that Notch inhibitor therapies could have potential side effects on muscle functions. STEM CELLS 2013;31:823–828

Role of LKB1-CRTC1 on Glycosylated COX-2 and Response to COX-2 Inhibition in Lung Cancer

BACKGROUND Cyclooxygenase-2 (COX-2) directs the synthesis of prostaglandins including PGE-2 linking inflammation with mitogenic signaling. COX-2 is also an anticancer target, however, treatment strategies have been limited by unreliable expression assays and by inconsistent tumor responses to COX-2 inhibition. METHODS We analyzed the TCGA and Directors Challenge lung cancer datasets (n = 188) and also generated an LKB1-null lung cancer gene signature (n = 53) to search the Broad Institute/Connectivity-MAP (C-MAP) dataset. We performed ChIP analyses, real-time polymerase chain reaction, immunoblotting, and drug testing of tumor cell lines (n = 8) and primary lung adenocarcinoma surgical resections (n = 13). RESULTS We show that COX-2 is a target of the cAMP/CREB coactivator CRTC1 signaling pathway. In addition, we detected a correlation between LKB1 status, CRTC1 activation, and presence of glycosylated, but not inactive hypoglycosylated COX-2 in primary lung adenocarcinoma. A search of the C-MAP drug database discovered that all high-ranking drugs positively associated with the LKB1-null signature are known CRTC1 activators, including forskolin and six different PGE-2 analogues. Somatic LKB1 mutations are present in 20.0% of lung adenocarcinomas, and we observed growth inhibition with COX-2 inhibitors in LKB1-null lung cancer cells with activated CRTC1 as compared with LKB1-wildtype cells (NS-398, P = .002 and Niflumic acid, P = .006; two-tailed t test). CONCLUSION CRTC1 activation is a key event that drives the LKB1-null mRNA signature in lung cancer. We also identified a positive feedback LKB1/CRTC1 signaling loop for COX-2/PGE2 regulation. These data suggest a role for LKB1 status and glycosylated COX-2 as specific biomarkers that provide a framework for selecting patients for COX-2 inhibition studies.

A Sensitive NanoString-Based Assay to Score STK11 (LKB1) Pathway Disruption in Lung Adenocarcinoma

Introduction: Serine/threonine kinase 11 gene (STK11), better known as liver kinase &bgr;1, is a tumor suppressor that is commonly mutated in lung adenocarcinoma (LUAD). Previous work has shown that mutational inactivation of the STK11 pathway may serve as a predictive biomarker for cancer treatments, including phenformin and cyclooxygenase‐2 inhibition. Although immunohistochemical (IHC) staining and diagnostic sequencing are used to measure STK11 pathway disruption, there are serious limitations to these methods, thus emphasizing the importance of validating a clinically useful assay. Methods: An initial STK11 mutation mRNA signature was generated using cell line data and refined using three large, independent patient databases. The signature was validated as a classifier using The Cancer Genome Atlas (TCGA) LUAD cohort as well as a 442‐patient LUAD cohort developed at Moffitt. Finally, the signature was adapted to a NanoString‐based format and validated using RNA samples isolated from formalin‐fixed, paraffin‐embedded tissue blocks corresponding to a cohort of 150 patients with LUAD. For comparison, STK11 IHC staining was also performed. Results: The STK11 signature was found to correlate with null mutations identified by exon sequencing in multiple cohorts using both microarray and NanoString formats. Although there was a statistically significant correlation between reduced STK11 protein expression by IHC staining and mutation status, the NanoString‐based assay showed superior overall performance, with a –0.1588 improvement in area under the curve in receiver‐operator characteristic curve analysis (p < 0.012). Conclusion: The described NanoString‐based STK11 assay is a sensitive biomarker to study emerging therapeutic modalities in clinical trials.

Abstract A22: An LKB1-CRTC1 circuit regulates glycosylated COX-2 and predicts drug response in lung cancer

Cyclooxygenase-2 (COX-2) directs the synthesis of prostaglandins important for mitogenic signaling. Here we report that COX-2 is a transcriptional target of the CREB co-activator CRTC1. In addition, we detected a correlation between the LKB1-null status and presence of 72/74 kDa glycosylated COX-2, but not inactive hypoglycosylated COX-2 in fresh lung adenocarcinoma samples. Since CRTC1 is suppressed by cytoplasmic shuttling following LKB1/AMPK/SIK phosphorylation, we developed an LKB1 signature in lung cancer to search the Connectivity-MAP drug response database. Remarkably, all high-ranking drugs positively associated with the LKB1-null signature were known CRTC1 activators. Somatic LKB1 mutations are present in 20% of lung adenocarcinomas and we observed growth and cell motility inhibition with COX-2 inhibitors in LKB1-null lung cancer cells with activated CRTC1, but negligible inhibition in LKB1-wildtype cells. In summary, the CREB co-activator CRTC family directly links LKB1 with COX-2 activation and provides a new framework for selecting patients for COX-2 inhibition. Citation Format: Chunxia Cao, Ruli Gao, Min Zhang, Antonio L. Amelio, Mohammad Fallahi, Zirong Chen, Yumei Gu, Chengbin Hu, Eric A. Welsh, Brienne E. Engel, Eric Haura, W. Douglas Cress, Lizi Wu, Maria Zajac-Kaye, Frederic J. Kaye. An LKB1-CRTC1 circuit regulates glycosylated COX-2 and predicts drug response in lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A22.

Abstract 2284: Generation and characterization of a mouse model of CRTC1-MAML2-induced mucoepidermoid carcinoma (MEC)

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy and can arise in multiple other organ sites. Currently, patients with advanced, unresectable MEC have limited therapeutic options and poor treatment outcomes. Clinical improvement has been hindered by a lack of understanding of the basic mechanisms underlying MEC development as well as suitable preclinical models. The majority of MEC tumors contain a t(11;19)(q21;p13) chromosomal translocation that results in the generation of a new fusion gene product, CRTC1-MAML2. We previously showed that the CRTC1-MAML2 fusion had a strong transcriptional co-activator activity and was capable of transforming epithelial cells in vitro, in part through co-activating the transcription factor CREB. Depletion of CRTC1-MAML2 fusion expression reduced the growth and survival of human malignant MEC cells when assayed in vitro or when propagated as xenograft tumors in vivo. These findings indicate that CRTC1-MAML2 is essential in maintaining MEC malignant phenotype and serves as a promising therapeutic target. However, whether CRTC1-MAML2 fusion has a causal role in MEC induction had not been demonstrated in vivo. In this study, we determined the oncogenic potential of the CRTC1-MAML2 fusion in vivo by establishing a Cre-regulated CRTC1-MAML2 transgenic mouse model. Through genetic crossing with MMTV-Cre mice or direct AAV-Cre transduction to induce expression of CRTC1-MAML2 transgene in salivary glandular cells, the transgenic mice developed salivary gland tumors with typical human MEC histological characteristics. Moreover, isolated tumor cells were capable of forming subcutaneous tumors in immune-compromised hosts that again recapitulate the MEC histological feature. Transcriptome analysis revealed that mouse tumors showed differential expression of genes associated with cell growth, survival, and metastasis as well as host cell immune modulating pathways in comparison with tumor-adjacent, macroscopically normal salivary gland tissues and salivary gland tissues from fusion transgene-negative littermate controls. Importantly, CRTC1-MAML2-induced tumors showed enhanced expression of known fusion target genes in human MEC, strongly supporting that this mouse tumor model molecularly resembles human MEC. Therefore, our study offered a direct proof for an oncogenic role of CRTC1-MAML2 fusion in vivo and provided the first genetically engineered mouse model for human MEC. Using this mouse MEC model, we are currently dissecting oncogenic transformation of normal salivary gland stem cells by salisphere assays, identifying cooperative genetic alterations in MEC through whole exome sequencing, and assessing its utility as a preclinical MEC model in evaluating therapeutic strategies. Citation Format: Zirong Chen, Jian-Liang Li, Shuibin Lin, Dinglong Pan, Wei Ni, Chunxia Cao, Yumei Gu, Maria Daniela Hurtado, Sergei Zolotukhin, Tao Sun, Frederic Kaye, Lizi Wu. Generation and characterization of a mouse model of CRTC1-MAML2-induced mucoepidermoid carcinoma (MEC). [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 2284. doi:10.1158/1538-7445.AM2015-2284

Abstract 166: Identification of a novel long noncoding RNA as a mediator for CRTC1-MAML2 fusion oncogenic function and a biomarker for CRTC1-MAML2 fusion-positive tumors

Long non-coding RNAs (LncRNA) are non-protein coding transcripts longer than 200 nucleotides and represent a novel class of gene regulators. LncRNA expression is frequently de-regulated in cancer. Specific lncRNAs have been shown to participate in cancer cell proliferation, survival, migration, and invasion, and targeting these cancer-associated lncRNAs inhibits tumor growth and metastasis. Therefore, lncRNAs are implicated as cancer biomarkers and therapeutic targets. The human genome encodes >10,000 lncRNAs; however, only a handful lncRNAs have been well characterized. We revealed through a microarray analysis that a novel lncRNA, lnc473 is a top target for the CRTC1-MAML2 fusion oncogene. CRTC1-MAML2 is generated by a t(11;19)(q21;p13) translocation that is specifically associated with mucoepidermoid carcinoma (MEC). MEC is a distinct type of tumors containing three cellular components, squamous cells, mucus-secreting cells, and intermediate cells. MEC occurs in many glandular tissues and accounts for the most common salivary gland malignancies. The CRTC1-MAML2 functions as a transcriptional co-activator, capable of promoting transcription from CREB and other factors. CRTC1-MAML2 is a major oncogenic driver for MEC initiation and maintenance. Currently, knowledge remains limited regarding how CRTC1-MAML2 mediates its oncogenic activity. In this study, we studied the regulatory mechanism of lnc473 expression and its function in mediating CRTC1-MAML2 oncogenic activity. We observed that lnc473 expression was tightly correlated with CRTC1-MAML2 fusion expression levels in human MEC cell lines and primary tumors using a Nanostring assay that allowed direct digital quantification of RNA molecules, strongly suggesting that lnc473 is a surrogate marker for functional CRTC1-MAML2 fusion. Lentiviral shRNA-mediated depletion of endogenous CRTCT1-MAML2 and its interacting transcription factor CREB resulted in a significant reduction of lnc473 expression. Also, lnc473 contained conserved CREB-binding sites on its proximal promoter, suggesting that CRTC1-MAML2 interacts with CREB in promoting lnc473 transcription. Functionally, depletion of lnc473 significantly reduced human MEC cell proliferation and survival in vitro and blocked the growth of MEC xenograft tumors. Our combined findings indicate that this lnc473 is a biomarker for human CRTC1-MAML2-positive MEC and has a novel regulatory function in mediating CRTC1-MAML2 cancer gene activity. Intriguingly, lnc473 depletion reduced expression of several known CRTC1-MAML2/CREB target genes, suggesting that lnc473 exerts a positive feedback regulation of CRTC1-MAML2-mediated transcription. The molecular mechanisms underlying lnc473 functions in MEC are currently being investigated through analyses of lnc473-regulated targeted gene profiling and lncRNA interacting protein complexes Citation Format: Zirong Chen, Jian-Liang Li, Shuibin Lin, Chunxia Cao, Frederic Kaye, Lizi Wu. Identification of a novel long noncoding RNA as a mediator for CRTC1-MAML2 fusion oncogenic function and a biomarker for CRTC1-MAML2 fusion-positive tumors. [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 166. doi:10.1158/1538-7445.AM2015-166

Abstract 591: LKB1 regulates COX-2 transcription via CRTCs dependent pathway

Inactivation of the liver kinase B1 (LKB1) gene by somatic mutation is a common event in lung adenocarcinoma. LKB1 was first identified as a tumor suppressor responsible for Peutz-Jeghers syndrome (PJS) and up regulation of cyclooxygenase-2 (COX-2) was detected in a significant percentage of human PJS polyps. In addition, COX-2 has been proposed as a therapeutic target for lung cancer although clinical trial using COX-2 inhibitors have been disappointing. In the current study, we identified a novel LKB1/CRTC/COX-2 molecular pathway in human lung cancer cells and provided new information for clinical use of COX-2 inhibitors. The Kaye lab cloned the CREB-regulated transcription co-activators (CRTCs) and showed aberrant phosphorylation and activation of CRTC1 exclusively in non-small cell lung cancer (NSCLC) samples carrying LKB1 null mutations. We have now demonstrated that CRTC directly regulates COX-2 expression via evolutionarily conserved CRE sites within the promoter. In addition, we observed that ectopic expression of LKB1 resulted in nuclear export of phosphorylated CRTC with attenuation of COX-2 expression. Alternatively, LKB1 knock-down results in CRTC activation and promotes COX-2 expression in NSCLC cells. Importantly, LKB1 null lung cancer cells were preferentially sensitive to COX-2 inhibitors. In summary, our data defines a CRTCs-mediated transcriptional regulatory mechanism for COX-2 activity in cancer and proposes a possible clinical application of COX-2 inhibitors in patients with lung tumors carrying somatic LKB1 null mutations. Citation Format: Chunxia Cao, Ruli Gao, Min Zhang, Zirong Chen, Lizi Wu, Maria Zajac Kaye, Frederic Kaye. LKB1 regulates COX-2 transcription via CRTCs dependent pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 591. doi:10.1158/1538-7445.AM2014-591

Abstract 3026: Targeting aberrantly activated AREG-EGFR signaling in CRTC1-MAML2-positive mucoepidermoid carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1-MAML2 gene fusion, encoded by a recurring chromosomal translocation t(11;19)(q14-21;p12-13), is a highly specific genetic alteration in more than 50% of mucoepidermoid carcinoma (MEC), the most common malignant SGT. In this study, we aimed to define the role of the CRTC1-MAML2 oncogene in the maintenance of MEC tumor growth and to investigate critical downstream target genes and pathways for therapeutic targeting of MEC. By performing gene expression analyses and functional studies via RNA interference and pharmacological modulation, we determined the importance of the CRTC1-MAML2 fusion gene and its downstream AREG-EGFR signaling in human MEC cancer cell growth ad survival in vitro and in vivo using human MEC xenograft models. We found that CRTC1-MAML2 fusion oncogene was required for the growth and survival of fusion-positive human MEC cancer cells in vitro and the in vivo. CRTC1-MAML2 induced the up-regulation of the EGFR ligand AREG by co-activating the transcription factor CREB and AREG subsequently activated EGFR signaling in an autocrine manner that promoted MEC cell growth and survival. Importantly, CRTC1-MAML2-positive MEC cells were highly sensitive to EGFR signaling inhibition. Therefore, our study revealed that aberrantly activated AREG-EGFR signaling is required for CRTC1-MAML2-positive MEC cell growth and survival, suggesting that EGFR-targeted therapies will benefit patients with unresectable CRTC1-MAML2-positive MEC. Citation Format: Zirong Chen, Yumei Gu, Chengbin Hu, Jian-Liang Li, Shuibin Lin, Huangxuan Shen, Chunxia Cao, Jie Chen, Jiancheng Li, Patick K. Ha, Federic J. Kaye, James D. Griffin, Lizi Wu. Targeting aberrantly activated AREG-EGFR signaling in CRTC1-MAML2-positive mucoepidermoid carcinoma. [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 3026. doi:10.1158/1538-7445.AM2013-3026