Lynn Vitale-Cross

Induction of ovarian cancer by defined multiple genetic changes in a mouse model system

We have developed a mouse model for ovarian carcinoma by using an avian retroviral gene delivery technique for the introduction of multiple genes into somatic ovarian cells of adult mice. Ovarian cells from transgenic mice engineered to express the gene encoding the avian receptor TVA were efficiently infected in vitro with multiple vectors carrying coding sequences for oncogenes and marker genes. When target cells were derived from TVA transgenic mice deficient for p53, the addition of any two of the oncogenes c-myc, K-ras, and Akt were sufficient to induce ovarian tumor formation when infected cells were injected at subcutaneous, intraperitoneal, or ovarian sites. We demonstrated that the ovarian surface epithelium is the precursor tissue for these ovarian carcinomas, and that introduction of oncogenes causes phenotypic changes in the ovarian surface epithelial cells. The induced ovarian tumors in mice resembled human ovarian carcinomas in their rapid progression and intraperitoneal metastatic spread.

Conditional Expression of K-ras in an Epithelial Compartment that Includes the Stem Cells Is Sufficient to Promote Squamous Cell Carcinogenesis

Ras genes are the most frequently mutated oncogenes in human cancer. However, the contribution of ras to tumor initiation still is unclear because ras expression in primary cells can cause cell cycle arrest and even cell death by apoptosis. Furthermore, when expressed in the epidermis of mice, mutant ras promotes the formation of benign papillomas, only few of which will progress into carcinomas. However, in these cases, ras-transgene expression often is restricted to suprabasal or follicular epithelial cells that may lack self-renewal capacity. Thus, it still is conceivable that expression of active ras in other epithelial compartments may exert a distinct ability to promote malignant progression. To address this possibility, transgenic mice carrying the tetracycline-inducible system (tet-on receptor) targeted to the basal layer of stratified epithelium, which includes the epithelial stem cells, were engineered and crossed with mice expressing the K-rasG12D oncogene under the control of tet-regulated responsive elements. On doxycycline administration, proliferative lesions ranging from hyperplasias, papillomas, and dysplasias to metastatic carcinomas developed in squamous epithelia of the skin, oral mucosa, salivary glands, tongue, esophagus, forestomach, and uterine cervix within just 10 to 20 days. The most noticeable lesions were invasive squamous carcinomas of the skin and oral mucosa. These findings suggest that the expression of oncogenes in an epithelial compartment that includes the stem cells may be sufficient to promote squamous carcinogenesis. They also provide a molecularly defined conditional animal model system in which the mechanisms responsible for cancer initiation, maintenance, and metastatic spread can be readily investigated.

Metformin Prevents the Development of Oral Squamous Cell Carcinomas from Carcinogen-Induced Premalignant Lesions

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. The recent identification of the mTOR complex 1 (mTORC1) signaling pathway as a highly prevalent molecular signature underlying HNSCC pathogenesis has provided the foundation to search for novel therapeutic approaches to prevent and treat HNSCC. Here, we asked whether metformin, the most widely used medication for the treatment of type II diabetes, which acts in part by stimulating the AMP-activated protein kinase (AMPK) signaling pathway thereby reducing mTORC1 activity, may lower the risk of HNSCC development. Indeed, we show that metformin reduces the growth of HNSCC cells and diminishes their mTORC1 activity by both AMPK-dependent and -independent mechanisms. We also optimized an oral-specific carcinogenesis mouse model that results in the accumulation of multiple oral premalignant lesions at the end of the carcinogen exposure, some of which then spontaneously progress into HNSCC. Using this mouse model, we observed that metformin specifically inhibits mTORC1 in the basal proliferating epithelial layer of oral premalignant lesions. Remarkably, metformin prevented the development of HNSCC by reducing significantly the size and number of carcinogen-induced oral tumoral lesions and by preventing their spontaneous conversion to squamous cell carcinomas. Collectively, our data underscore the potential clinical benefits of using metformin as a targeted chemopreventive agent in the control of HNSCC development and progression. Cancer Prev Res; 5(4); 562–73. ©2012 AACR.

Chemical Carcinogenesis Models for Evaluating Molecular-Targeted Prevention and Treatment of Oral Cancer

Editors Note: Cancer Prevention Research is dedicated to presenting leading-edge work in cancer prevention-related models of carcinogenesis. This minireview details the evolution and development of a mouse model of 4-nitroquinoline-1 oxide (4NQO)-induced oral carcinogenesis, thus extending

mTOR Co-Targeting in Cetuximab Resistance in Head and Neck Cancers Harboring PIK3CA and RAS Mutations

Background Cetuximab, a monoclonal blocking antibody against the epidermal growth factor receptor EGFR, has been approved for the treatment of squamous cell carcinomas of the head and neck (HNSCC). However, only few patients display long-term responses, prompting the search for cetuximab resistance mechanisms and new therapeutic options enhancing cetuximab effectiveness. Methods Cetuximab-sensitive HNSCC cells were retro-engineered to express PIK3CA and RAS oncogenes. These cells and HNSCC cells harboring endogenous PIK3CA and RAS oncogenes were xenografted into mice (n = 10 per group) and studied for their biochemical, antitumor, antiangiogenic, and antilymphangiogenic responses to cetuximab and mTOR targeting agents. All P values are two-sided. Results Cetuximab treatment of PIK3CA- and RAS-expressing HNSCC xenografts promoted an initial antitumor response, but all tumors relapsed within few weeks. In these tumors, cetuximab did not decrease the activity of mTOR, a downstream signaling target of EGFR, PIK3CA, and RAS. The combined administration of cetuximab and mTOR inhibitors exerted a remarkably increased antitumor activity, particularly in HNSCC cells that are resistant to cetuximab as a single agent. Indeed, cotargeting mTOR together with cetuximab caused a rapid tumor collapse of both PIK3CA- and RAS-expressing HNSCC xenografts (P < .001), concomitant with reduced proliferation (P < .001) and lymphangiogenesis (P < .001). Conclusion The presence of PIK3CA and RAS mutations and other alterations affecting the mTOR pathway activity in HNSCC could be exploited to predict the potential resistance to cetuximab, and to select the patients that may benefit the most from the concomitant administration of cetuximab and PI3K and/or mTOR inhibitors as a precision molecular therapeutic option for HNSCC patients.

Prevention of tumor growth driven by PIK3CA and HPV oncogenes by targeting mTOR signaling with metformin in oral squamous carcinomas expressing OCT3

Most squamous cell carcinomas of the head and neck (HNSCC) exhibit a persistent activation of the PI3K–mTOR signaling pathway. We have recently shown that metformin, an oral antidiabetic drug that is also used to treat lipodystrophy in HIV-infected (HIV+) individuals, diminishes mTOR activity and prevents the progression of chemically induced experimental HNSCC premalignant lesions. Here, we explored the preclinical activity of metformin in HNSCCs harboring PIK3CA mutations and HPV oncogenes, both representing frequent HNSCC alterations, aimed at developing effective targeted preventive strategies. The biochemical and biologic effects of metformin were evaluated in representative HNSCC cells expressing mutated PIK3CA or HPV oncogenes (HPV+). The oral delivery of metformin was optimized to achieve clinical relevant blood levels. Molecular determinants of metformin sensitivity were also investigated, and their expression levels were examined in a large collection of HNSCC cases. We found that metformin inhibits mTOR signaling and tumor growth in HNSCC cells expressing mutated PIK3CA and HPV oncogenes, and that these activities require the expression of organic cation transporter 3 (OCT3/SLC22A3), a metformin uptake transporter. Coexpression of OCT3 and the mTOR pathway activation marker pS6 were observed in most HNSCC cases, including those arising in HIV+ patients. Activation of the PI3K–mTOR pathway is a widespread event in HNSCC, including HPV− and HPV+ lesions arising in HIV+ patients, all of which coexpress OCT3. These observations may provide a rationale for the clinical evaluation of metformin to halt HNSCC development from precancerous lesions, including in HIV+ individuals at risk of developing HPV− associated cancers. Cancer Prev Res; 8(3); 197–207. ©2015 AACR.

Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo.

Liver kinase B1 (LKB1) and its downstream effector AMP‐activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich‐cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver‐specific (albumin‐Cre) LKB1 knockout mice (LKB1−/−) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1−/− livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6‐carboxyfluorescein diacetate (6‐CFDA), which is processed in hepatocytes into its fluorescent derivative 6‐carboxyfluorescein (6‐CF) and secreted into the canaliculi. In LKB1−/− mice, 6‐CF remained largely in hepatocytes, canalicular secretion was delayed, and 6‐CF appeared in the blood. To test whether 6‐CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6‐CFDA. In wild‐type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1−/− mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. Conclusion: LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317‐1329)

LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.

Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.

Vasopressin stimulates the proliferation and differentiation of red blood cell precursors and improves recovery from anemia

Vasopressin stimulates erythropoiesis. The body’s stop-gap solution for anemia Vasopressin is an antidiuretic hormone, whose best known functions are to promote water retention and maintain fluid balance. However, it also has other effects, and Mayer et al. discovered that these include stimulation of red blood cell maturation. By studying human patients with central diabetes insipidus (vasopressin deficiency) and a rat model that lacks vasopressin, the authors determined that this hormone acts in concert with erythropoietin but on a shorter time scale. Erythropoietin stimulates red blood cell production starting with early progenitors, which takes time, whereas vasopressin promotes maturation of intermediate red blood cell precursors and thus allows rapid recovery after blood loss while the new cells stimulated by erythropoietin are still maturing. Arginine vasopressin (AVP) made by hypothalamic neurons is released into the circulation to stimulate water resorption by the kidneys and restore water balance after blood loss. Patients who lack this antidiuretic hormone suffer from central diabetes insipidus. We observed that many of these patients were anemic and asked whether AVP might play a role in red blood cell (RBC) production. We found that all three AVP receptors are expressed in human and mouse hematopoietic stem and progenitor cells. The AVPR1B appears to play the most important role in regulating erythropoiesis in both human and mouse cells. AVP increases phosphorylation of signal transducer and activator of transcription 5, as erythropoietin (EPO) does. After sublethal irradiation, AVP-deficient Brattleboro rats showed delayed recovery of RBC numbers compared to control rats. In mouse models of anemia (induced by bleeding, irradiation, or increased destruction of circulating RBCs), AVP increased the number of circulating RBCs independently of EPO. In these models, AVP appears to jump-start peripheral blood cell replenishment until EPO can take over. We suggest that specific AVPR1B agonists might be used to induce fast RBC production after bleeding, drug toxicity, or chemotherapy.

Assembly and activation of the Hippo signalome by FAT1 tumor suppressor

Dysregulation of the Hippo signaling pathway and the consequent YAP1 activation is a frequent event in human malignancies, yet the underlying molecular mechanisms are still poorly understood. A pancancer analysis of core Hippo kinases and their candidate regulating molecules revealed few alterations in the canonical Hippo pathway, but very frequent genetic alterations in the FAT family of atypical cadherins. By focusing on head and neck squamous cell carcinoma (HNSCC), which displays frequent FAT1 alterations (29.8%), we provide evidence that FAT1 functional loss results in YAP1 activation. Mechanistically, we found that FAT1 assembles a multimeric Hippo signaling complex (signalome), resulting in activation of core Hippo kinases by TAOKs and consequent YAP1 inactivation. We also show that unrestrained YAP1 acts as an oncogenic driver in HNSCC, and that targeting YAP1 may represent an attractive precision therapeutic option for cancers harboring genomic alterations in the FAT1 tumor suppressor genes.Dysregulation of the Hippo signaling is a frequent event in human malignancies, but the molecular mechanisms remain unclear. Here the authors show that in head and neck squamous carcinoma, FAT1 interacts with the Hippo signaling complex, resulting in the activation of core Hippo kinases and YAP1 inactivation.