GNAQ and GNA11 are heterotrimeric G protein alpha subunits which are mutated in a mutually exclusive pattern in most cases of uveal melanoma, one of the most aggressive cancers. Here we introduce the first transgenic mouse model of uveal melanoma, which develops cancers induced by expression of oncogenic GNAQ Q209L under control of the Rosa26 promoter. Disease penetrance is 100% by 3 months of age with 94% of mice also developing lung tumors. In this model, the Yap protein of the Hippo pathway is activated in the eyes, and blood vessels near the lesions in the head and lungs exhibit melanocytic invasion. While full transcription levels are not necessary for GNAQ Q209L to transform mouse melanocytes, we obtained suggestive evidence of a selective advantage for increased GNAQ Q209L expression in human tumors. Intriguingly, enforced expression of GNAQ Q209L progressively eliminated melanocytes from the inter-follicular epidermis in adults, possibly explaining the near absence of GNAQ Q209 mutations in human epithelial melanomas. The mouse model also exhibited dermal nevi and melanocytic neoplasia of the central nervous system, accompanied by impaired hearing and balance, identifying a novel role for GNAQ in melanocyte-like cells of the inner ear. Overall, this model offers a new tool to dissect signaling by oncogenic GNAQ and test potential therapeutics, in an in vivo setting where GNAQ Q209L mutations contribute to both the initiation and metastatic progression of uveal melanoma.

Pancreatic ductal adenocarcinoma (PDAC) develops via an accumulation of various gene mutations. The mechanism underlying the mutations in PDAC development, however, is not fully understood. Recent insight into the close association between the mutation pattern of various cancers and specific mutagens led us to investigate the possible involvement of activation-induced cytidine deaminase (AID), a DNA editing enzyme, in pancreatic tumorigenesis. Our immunohistochemical findings revealed AID protein expression in human acinar ductal metaplasia, pancreatic intraepithelial neoplasia, and PDAC. Both the amount and intensity of the AID protein expression increased with the progression from precancerous to cancerous lesions in human PDAC tissues. To further assess the significance of ectopic epithelial AID expression in pancreatic tumorigenesis, we analyzed the phenotype of AID transgenic (AID Tg) mice. Consistent with our hypothesis that AID is involved in the mechanism of the mutations underlying pancreatic tumorigenesis, we found precancerous lesions developing in the pancreas of AID Tg mice. Using deep sequencing, we also detected Kras and c-Myc mutations in our analysis of the whole pancreas of AID Tg mice. In addition, Sanger sequencing confirmed the presence of Kras, c-Myc, and Smad4 mutations, with the typical mutational footprint of AID in precancerous lesions in AID Tg mice separated by laser capture microdissection. Taken together, our findings suggest that AID contributes to the development of pancreatic precancerous lesions by inducing tumor-related gene mutations. Our new mouse model without intentional manipulation of specific tumor-related genes provides a powerful system for analyzing the mutations involved in PDAC. Cancer Res; 75(16); 1–10. ©2015 AACR.

Pancreatic ductal adenocarcinoma (PDA) develops via an accumulation of various gene mutations. The mechanism underlying the mutations in PDA development, however, is not fully understood. Recent insight into the close association between the mutation pattern of various cancers and specific mutagens led us to investigate the possible involvement of activation-induced cytidine deaminase (AID), a DNA editing enzyme, in pancreatic tumorigenesis. Our immunohistochemistry findings revealed AID protein expression in human acinar ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN), and PDA. Both the amount and intensity of the AID protein expression increased with the progression from precancerous to cancerous lesions in human PDA tissues. To further assess the significance of ectopic epithelial AID expression in pancreatic tumorigenesis, we analyzed the phenotype of AID transgenic (AID Tg) mice. Consistent with our hypothesis that AID is involved in the mechanism of the mutations underlying pancreatic tumorigenesis, we found precancerous lesions developing in the pancreas of AID Tg mice. Using deep sequencing, we also detected Kras and c-Myc mutations in our analysis of the whole pancreas of AID Tg mice. In addition, Sanger sequencing confirmed the presence of Kras, c-Myc and Smad4 mutations with the typical mutational footprint of AID in precancerous lesions in AID Tg mice separated by laser capture microdissection. Taken together, our findings suggest that AID contributes to the development of pancreatic precancerous lesions by inducing tumor-related gene mutations. Our new mouse model without intentional manipulation of specific tumor-related genes provides a powerful system for analyzing the mutations involved in PDA.

Keratins that are overexpressed selectively in human carcinomas may offer diagnostic and prognostic utility. In this study, we show that high expression of keratin-17 (K17) predicts poor outcome in patients with cervical cancer, at early or late stages of disease, surpassing in accuracy either tumor staging or loss of p27KIP1 as a negative prognostic marker in this setting. We investigated the mechanistic basis for the biologic impact of K17 through loss- and gain-of-function experiments in human cervix, breast, and pancreatic cancer cells. Specifically, we determined that K17 functions as an oncoprotein by regulating the subcellular localization and degradation of p27KIP1. We found that K17 was released from intermediate filaments and translocated into the nucleus via a nuclear localization signal (NLS), specific among keratins, where it bound p27KIP1 during G1 phase of the cell cycle. p27KIP1 lacks a nuclear export signal (NES) and requires an adaptor for CRM1 binding for nuclear export. In K17, we defined and validated a leucine-rich NES that mediated CRM1 binding for export. Cervical cancer cells expressing K17 mutations in its NLS or NES signals exhibited an increase in levels of nuclear p27KIP1, whereas cells expressing wild-type K17 exhibited a depletion in total endogenous p27KIP1. In clinical specimens of cervical cancer, we confirmed that the expressions of K17 and p27KIP1 were inversely correlated, both across tumors and within individual tumors. Overall, our findings establish that K17 functions specially among keratins as an oncoprotein by controlling the ability of p27KIP1 to influence cervical cancer pathogenesis. Cancer Res; 75(17); 1–13. ©2015 AACR.

Keratins that are overexpressed selectively in human carcinomas may offer diagnostic and prognostic utility. In this study, we show how high expression of keratin-17 (K17) predicts poor outcome in cervical cancer patients, at early or late stages of disease, surpassing in accuracy either tumor staging or loss of p27KIP1 as a negative prognostic marker in this setting. We investigated the mechanistic basis for the biological impact of K17 through loss and gain of function experiments in human cervix, breast and pancreatic cancer cells. Specifically, we determined that K17 functions as an oncoprotein by regulating the subcellular localization and degradation of p27KIP1. We found that K17 was released from intermediate filaments and translocated into the nucleus via a nuclear localization signal (NLS), specific among keratins, where it bound p27KIP1 during G1 phase of the cell cycle. p27KIP1 lacks a nuclear export signal (NES) and requires an adaptor for CRM1 binding for nuclear export. In K17 we defined and validated a leucine-rich NES that mediated CRM1 binding for export. Cervical cancer cells expressing K17 mutations in its NLS or NES signals exhibited an increase in levels of nuclear p27KIP1, whereas cells expressing wild-type K17 exhibited a depletion in total endogenous p27KIP1. In clinical specimens of cervical cancer, we confirmed that the expression of K17 and p27KIP1 were inversely correlated, both across tumors and within individual tumors. Overall, our findings establish that K17 functions specially among keratins as an oncoprotein, by controlling the ability of p27KIP1 to influence cervical cancer pathogenesis.