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

Bypassing estrogen receptor (ER) signaling during development of endocrine resistance remains the most common cause of disease progression and mortality in breast cancer patients. To date, the majority of molecular research on ER action in breast cancer has occurred in cell line models derived from late stage disease. Here we describe patient-derived ER + luminal breast tumor models for the study of intratumoral hormone and receptor action. Human breast tumor samples obtained from patients post surgery were immediately transplanted into NOD/SCID or NOD/SCID/ILIIrg−/− mice under estrogen supplementation. Five transplantable patient-derived ER + breast cancer xenografts were established, derived from both primary and metastatic cases. These were assessed for estrogen dependency, steroid receptor expression, cancer stem cell content, and endocrine therapy response. Gene expression patterns were determined in select tumors ±estrogen and ±endocrine therapy. Xenografts morphologically resembled the patient tumors of origin, and expressed similar levels of ER (5–99 %), and progesterone and androgen receptors, over multiple passages. Four of the tumor xenografts were estrogen dependent, and tamoxifen or estrogen withdrawal (EWD) treatment abrogated estrogen-dependent growth and/or tumor morphology. Analysis of the ER transcriptome in select tumors revealed notable differences in ER mechanism of action, and downstream activated signaling networks, in addition to identifying a small set of common estrogen-regulated genes. Treatment of a na¨ıve tumor with tamoxifen or EWD showed similar phenotypic responses, but relatively few similarities in estrogen-dependent transcription, and affected signaling pathways. Several core estrogen centric genes were shared with traditional cell line models. However, novel tumor-specific estrogen-regulated potential target genes, such as cancer/testis antigen 45, were uncovered. These results evoke the importance of mapping both conserved and tumor-unique ER programs in breast cancers. Furthermore, they underscore the importance of primary xenografts for improved understanding of ER+ breast cancer heterogeneity and development of personalized therapies.

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease.

Summary Genomic rearrangements resulting in activating kinase fusions have been increasingly described in a number of cancers including malignant melanoma, but their frequency in specific melanoma subtypes has not been reported. We used break-apart fluorescence in-situ hybridization (FISH) to identify genomic rearrangements in tissues from 59 patients with various types of malignant melanoma including acral lentiginous, mucosal, superficial spreading, and nodular. We identified four genomic rearrangements involving the genes BRAF, RET, and ROS1. Of these, three were confirmed by IHC or sequencing and one was found to be an ARMC10-BRAF fusion that has not been previously reported in melanoma. These fusions occurred in different subtypes of melanoma but all in tumors lacking known driver mutations. Our data suggest gene fusions are more common than previously thought-and should be further explored particularly in melanomas lacking known driver mutations.