Inhibition of protein phosphatase 2A (PP2A) activity has been identified as a prerequisite for the transformation of human cells. However, the molecular mechanisms by which PP2A activity is inhibited in human cancers are currently unclear. In this study, we describe a cellular inhibitor of PP2A with oncogenic activity. The protein, designated Cancerous Inhibitor of PP2A (CIP2A), interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage-independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in two common human malignancies, head and neck squamous cell carcinoma (HNSCC) and colon cancer. Thus, our data show that CIP2A is a human oncoprotein that inhibits PP2A and stabilizes c-Myc in human malignancies.

Smad4 is a central mediator of TGF-β signaling, and its expression is downregulated or lost at the malignant stage in several cancer types. In this study, we found that Smad4 was frequently downregulated not only in human head and neck squamous cell carcinoma (HNSCC) malignant lesions, but also in grossly normal adjacent buccal mucosa. To gain insight into the importance of this observation, we generated mice in which Smad4 was deleted in head and neck epithelia (referred to herein as HN-Smad4 -/-mice) and found that they developed spontaneous HNSCC. Interestingly, both normal head and neck tissue and HNSCC from HN-Smad4 -/-mice exhibited increased genomic instability, which correlated with downregulated expression and function of genes encoding proteins in the Fanconi anemia/Brca (Fanc/Brca) DNA repair pathway linked to HNSCC susceptibility in humans. Consistent with this, further analysis revealed a correlation between downregulation of Smad4 protein and downregulation of the Brca1 and Rad51 proteins in human HNSCC. In addition to the above changes in tumor epithelia, both normal head and neck tissue and HNSCC from HN-Smad4 -/-mice exhibited severe inflammation, which was associated with increased expression of TGF-β1 and activated Smad3. We present what we believe to be the first single gene-knockout model for HNSCC, in which both HNSCC formation and invasion occurred as a result of Smad4 deletion. Our results reveal an intriguing connection between Smad4 and the Fanc/Brca pathway and highlight the impact of epithelial Smad4 loss on inflammation.

The prognosis of head-and-neck squamous cell carcinoma (HNSCC) has not been improved in the past 20 years. Validation of HNSCC biomarkers for targeted therapy has been hindered by a lack of animal models mimicking human HNSCC at both the pathological and molecular levels. Here we report that overexpression of K-ras or H-ras and loss of transforming growth factor-␤ type II receptor (TGF␤RII) are common events in human HNSCC. Activation of either K-ras or H-ras in combination with TGF␤RII deletion from mouse head-and-neck epithelia caused HNSCC with complete penetrance, some of which progressed to metastases. These tumors displayed pathology indistinguishable from human HNSCCs and exhibited multiple molecular alterations commonly found in human HNSCCs. Additionally, elevated endogenous TGF␤1 in these lesions contributed to inflammation and angiogenesis. Our data suggest that targeting common oncogenic pathways in tumor epithelia together with blocking the effect of TGF␤1 on tumor stroma may provide a novel therapeutic strategy for HNSCC.[Keywords: HNSCC; head-and-neck-specific knockout; metastasis; Ras; TGF␤RII; TGF␤1] Supplemental material is available at http://www.genesdev.org.

TGF-β and its signaling mediators, Smad2, -3, and -4, are involved with tumor suppression and promotion functions. Smad4 -/-mouse epidermis develops spontaneous skin squamous cell carcinomas (SCCs), and Smad3 -/-mice are resistant to carcinogen-induced skin cancer; however, the role of Smad2 in skin carcinogenesis has not been explored. In the present study, we found that Smad2 and Smad4, but not Smad3, were frequently lost in human SCCs. Mice with keratinocyte-specific Smad2 deletion exhibited accelerated formation and malignant progression of chemically induced skin tumors compared with WT mice. Consistent with the loss of Smad2 in poorly differentiated human SCCs, Smad2 -/-tumors were poorly differentiated and underwent epithelial-mesenchymal transition (EMT) prior to spontaneous Smad4 loss. Reduced E-cadherin and activation of its transcriptional repressor Snail were also found in Smad2 -/-mouse epidermis and occurred more frequently in Smad2-negative human SCCs than in Smad2-positive SCCs. Knocking down Snail abrogated Smad2 loss-associated EMT, suggesting that Snail upregulation is a major mediator of Smad2 loss-associated EMT. Furthermore, Smad2 loss led to a significant increase in Smad4 binding to the Snail promoter, and knocking down either Smad3 or Smad4 in keratinocytes abrogated Smad2 loss-associated Snail overexpression. Our data suggest that enhanced Smad3/Smad4-mediated Snail transcription contributed to Smad2 loss-associated EMT during skin carcinogenesis.

In the present study, we show that transforming growth factor ␤1 (TGF-␤1) was frequently overexpressed in human head and neck squamous cell carcinomas (HNSCCs) and adjacent tissues in comparison with normal head and neck tissues. To determine the role of TGF-␤1 overexpression in HNSCC carcinogenesis, we generated transgenic mice in which TGF-␤1 transgene expression can be induced in head and neck epithelia. TGF-␤1 transgene induction in head and neck epithelia, at levels similar to those in human HNSCCs, caused severe inflammation and angiogenesis. Consequently, TGF-␤1-transgenic epithelia exhibited hyperproliferation. These phenotypes correlated with enhanced Smad signaling in transgenic epithelia and stroma. Our study suggests that TGF-␤1 overexpression at early stages of HNSCC formation provides a tumor promoting microenvironment.

Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene Kras G12D activation and Smad4 deletion, to mouse keratin 15-expressing (K15 + ) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell-enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9-transduced human SCC cells exhibited increased invasion. We identified α-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with α-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with Kras G12D activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs. IntroductionSquamous cell carcinomas (SCCs) are derived from stratified epithelia present within the skin and oral cavity. A subset of aggressive SCCs become metastatic and lead to metastasis-associated death. The rate of metastasis in skin SCCs ranges from 0.1% to 10% (1), with poorly differentiated tumors and those with greater vertical tumor thickness having an increased risk of metastasis (2). Genetic alterations and intrinsic tumor cell properties controlling SCC metastasis are largely unknown. Genetically engineered mice provide a powerful tool for dissecting driver mutations that contribute to SCC initiation and metastasis. To date, very few genetic mutations causing spontaneous SCC formation and metastasis have been found, particularly metastasis to the lung, which is the leading cause of SCC-associated death (3). Mice with a Smad4 deletion in stratified epithelia develop spontaneous SCCs in the skin, oral cavity, and forestomach (4-6). Among these models, oral SCCs metastasize to lymph nodes (4), whereas skin and forestomach SCCs do not metastasize (5, 6).Because stratified epithelia undergo constant self-renewal and rapid turnover, it is believed that driver mutations for SCCs must initially occur in resident stem cells that renew these epithelia throughout life. In mouse skin, the hair follicle bulge harbors

summary Detection of DNA methylation has produced promising results as biomarkers for head and neck squamous cell carcinoma (HNSCC). However, current panels are limited by an insufficient number of sensitive and specific tumor markers. MicroRNAs (miR) play an important role in tumorigenesis, and may represent a novel panel of molecules for the development of cancer biomarkers. We investigated methylation of three miRNA promoter sites of miR-9 (miR-9-1, miR-9-2, miR-9-3) in 107 human head and neck tissue samples and controls. We found methylations of miR-9-1 and miR-9-3 were higher in oral and oropharyngeal carcinomas than that in laryngeal carcinoma, achieving a combined sensitivity of 63% and 56%, respectively, for these two tumor types, compared to 21% for the laryngeal carcinoma. Quantitative PCR of miR-9 showed reduced expression associated with methylation of miR-9 in tumor tissues. To investigate the functional consequences of miR-9 methylation, we found that miR-9 methylation is correlated with miR-9 expression level in human HNSCC cell lines. Demethylation treatment using 5-aza-deoxycytidine restored its expression in a miR-9 methylated human HNSCC cell line UM-SCC22A. Furthermore, cell proliferation and viability was significantly inhibited, while PTEN expression was elevated after transfection of miR-9 into the UM-SCC22A cell line. In summary, our results suggest that methylations of miR-9-1 and miR-9-3 are sensitive and specific biomarkers for HNSCC, particularly for oral and oropharyngeal squamous cell carcinomas. In addition, miR-9 may function as a tumor suppressor in HNSCC through inhibition of cell proliferation and elevation of tumor suppressor PTEN.

BACKGROUND Endometriosis is frequently associated with and thought of having propensity to develop into ovarian clear cell carcinoma (OCCC), although the molecular transformation mechanism is not completely understood. METHODS We employed immunohistochemical (IHC) staining for marker expression along the potential progression continuum. Expression profiling of microdissected endometriotic and OCCC cells from patient-matched formalin-fixed, paraffin-embedded samples was performed to explore the carcinogenic pathways. Function of novel biomarkers was confirmed by knockdown experiments. RESULTS PTEN was significantly lost in both endometriosis and invasive tumor tissues, while estrogen receptor (ER) expression was lost in OCCC relative to endometriosis. XRCC5, PTCH2, eEF1A2, and PPP1R14B were significantly overexpressed in OCCC and associated endometriosis, but not in benign endometriosis (p≤0.004). Knockdown experiments with XRCC5 and PTCH2 in a clear cell cancer cell line resulted in significant growth inhibition. There was also significant silencing of a panel of target genes with histone H3 lysine 27 trimethylation, a signature of polycomb chromatin-remodeling complex in OCCC. IHC confirmed the loss of expression of one such polycomb target gene, the serous ovarian cancer lineage marker WT1 in OCCC, while endometriotic tissues showed significant co-expression of WT1 and ER. CONCLUSIONS Loss of PTEN expression is proposed as an early and permissive event in endometriosis development, while the loss of ER and polycomb-mediated transcriptional reprogramming for pluripotency may play an important role in the ultimate transformation process. Our study provides new evidence to redefine the pathogenic program for lineage-specific transformation of endometriosis to OCCC.