Mutational activation of BRAF is the earliest and most common genetic alteration in human melanoma. Hence, to build a model of human melanoma, we generated mice with conditional melanocyte-specific expression of BRafV600E. Upon induction of BRafV600E expression, mice developed benign melanocytic hyperplasias that failed to progress to melanoma over 15-20 months. By contrast, expression of BRafV600E combined with Pten tumor suppressor gene silencing elicited development of melanoma with 100% penetrance, short latency and with metastases observed in lymph nodes and lungs. Melanoma was prevented by inhibitors of mTorc1 (Rapamycin) or MEK1/2 (PD325901) but, upon cessation of drug administration, mice developed melanoma indicating the presence of long-lived melanoma-initiating cells in this system. Importantly, combined treatment with Rapamycin and PD325901 led to shrinkage of established melanomas. These mice, engineered with a common genetic profile to human melanoma, provide an excellent system to study melanoma’s cardinal feature of metastasis and for pre-clinical evaluation of agents designed to prevent or treat metastatic disease.

Mutationally activated BRAF V600E (BRAF VE) is detected in ∼6% of human malignancies and promotes sustained MEK1/2-ERK1/2 pathway activation. We have designed BRaf CA mice to express normal BRaf prior to Cre-mediated recombination after which BRaf VE is expressed at physiological levels. BRaf CA mice infected with an Adenovirus expressing Cre recombinase developed benign lung tumors that only rarely progressed to adenocarcinoma. Moreover, BRaf VE -induced lung tumors were prevented by pharmacological inhibition of MEK1/2. BRaf VE expression initially induced proliferation that was followed by growth arrest bearing certain hallmarks of senescence. Consistent with Ink4a/Arf and TP53 tumor suppressor function, BRaf VE expression combined with mutation of either locus led to cancer progression.Supplemental material is available at http://www.genesdev.org.

SUMMARY Malignant melanoma is characterized by frequent metastasis, however specific changes that regulate this process have not been clearly delineated. Although it is well known that Wnt signaling is frequently dysregulated in melanoma, the functional implications of this observation are unclear. By modulating β-catenin levels in a mouse model of melanoma that is based on melanocyte-specific Pten loss and BrafV600E mutation, we demonstrate that β-catenin is a central mediator of melanoma metastasis to lymph node and lung. In addition to altering metastasis, β-catenin levels control tumor differentiation and regulate both MAPK/Erk and PI3K/Akt signaling. Highly metastatic tumors with β-catenin stabilization are very similar to a subset of human melanomas. Together these findings establish Wnt signaling as a metastasis regulator in melanoma.

Human melanocytic nevi (moles) are benign lesions harboring activated oncogenes, including BRAF. Although this oncogene initially acts mitogenically, eventually, oncogene-induced senescence (OIS) ensues. Nevi can infrequently progress to melanomas, but the mechanistic relationship with OIS is unclear. We show here that PTEN depletion abrogates BRAF V600E -induced senescence in human fibroblasts and melanocytes. Correspondingly, in established murine BRAF V600E -driven nevi, acute shRNA-mediated depletion of PTEN prompted tumor progression. Furthermore, genetic analysis of laser-guided microdissected human contiguous nevus-melanoma specimens recurrently revealed identical mutations in BRAF or NRAS in adjacent benign and malignant melanocytes. The PI3K pathway was often activated through either decreased PTEN or increased AKT3 expression in melanomas relative to their adjacent nevi. Pharmacologic PI3K inhibition in melanoma cells suppressed proliferation and induced the senescence-associated tumor suppressor p15INK4B . This treatment also eliminated subpopulations resistant to targeted BRAF V600E inhibition. Our findings suggest that a significant proportion of melanomas arise from nevi. Furthermore, these results demonstrate that PI3K pathway activation serves as a rate-limiting event in this setting, acting at least in part by abrogating OIS. The reactivation of senescence features and elimination of cells refractory to BRAF V600E inhibition by PI3K inhibition warrants further investigation into the therapeutic potential of simultaneously targeting these pathways in melanoma.

KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDA), but remains an intractable pharmacological target. Consequently, defining RAS effector pathway(s) required for PDA initiation and maintenance is critical to improve treatment of this disease. Here we demonstrate that expression of BRAFV600E, but not PIK3CAH1047R, in the mouse pancreas leads to pancreatic intraepithelial neoplasia (PanIN) lesions. Moreover, concomitant expression of BRAFV600E and TP53R270H result in lethal PDA. We tested pharmacologic inhibitors of Ras effectors against multiple human PDA cell lines. MEK inhibition was highly effective both in vivo and in vitro, and was synergistic with AKT inhibition in most cell lines tested. We demonstrate that RAF→MEK→ERK signaling is central to the initiation and maintenance of PDA and to rational combination strategies in this disease. These results emphasize the value of leveraging multiple complementary experimental systems to prioritize pathways for effective intervention strategies in PDA.

Amplification of the Neu/c-erbB-2 receptor tyrosine kinase has been implicated as an important event in the genesis of human breast cancer. Indeed, transgenic mice bearing either an activated form of neu or the wild-type proto-oncogene under the transcriptional control of the mouse mammary tumor virus promoterenhancer frequently develop mammary carcinomas (L. Bouchard, L. Lamarre, P. J. Tremblay, and P. Jolicoeur, Cell 57:931-936, 1989 Leder, Cell 54:105-115, 1988). Induction of mammary tumors in transgenic mice expressing the wild-type Neu receptor is associated with activation of the receptor's intrinsic tyrosine kinase activity (Guy et al., Proc. Natl. Acad. Sci. USA 89:10578-10582, 1992). Here, we demonstrate that activation of Neu in these transgenic mice occurs through somatic mutations located within the transgene itself. Sequence analyses of these mutations revealed that they contain in-frame deletions of 7 to 12 amino acids in the extracellular region proximal to the transmembrane domain. Introduction of these mutations into a wild-type neu cDNA results in an increased transforming ability of the altered Neu tyrosine kinase. These observations suggest that oncogenic activation of Neu in mammary tumorigenesis frequently occurs by somatic mutation.The neu (c-erbB-2 or HER2) proto-oncogene encodes a receptor tyrosine kinase belonging to the epidermal growth factor receptor family (2, 9). Oncogenic activation of Neu can occur through multiple molecular mechanisms, including a point mutation in its transmembrane domain (3) and deletion of the extracellular domain (4). Amplification and consequent overexpression of neulc-erbB-2 have also been observed in a significant proportion of human breast and ovarian cancers (14,24,27). In fact, several studies have shown that the degree of amplification is inversely correlated to a poor clinical outcome for breast cancer patients in whom the cancer has not spread to the lymph nodes (11,22). Although elevated levels of neulc-erbB-2 are observed in these human cancers, no comparable mutations have been detected in these tumor samples (15).Consistent with these clinical observations, expression of activated or wild-type neu targeted to the mammary epithelia of transgenic mice results in the efficient induction of mammary tumors (5,12,19). In mice expressing elevated levels of the wild-type Neu protein, the appearance of mammary tumors is associated with a high incidence of metastatic disease (12). Mammary gland-specific expression of Neu results in the appearance of focal mammary tumors after a variable latency period (12). Both the focal nature and long latency period suggest that additional genetic events are needed to transform the mammary epithelium. Mammary tumorigenesis in these transgenic strains is correlated with elevated intrinsic Neu tyrosine kinase activity and the de novo tyrosine phosphorylation of several cellular proteins (12). The RNA and protein (12).While it is clear that activation of Neu plays an important role in the induction of mammary tumors...

Clinical studies have revealed that cancer patients whose tumours have increased ErbB2 expression tend to have more aggressive, metastatic disease, which is associated with parameters predicting a poor outcome. The molecular basis underlying ErbB2-dependent cell motility and metastases formation, however, still remains poorly understood. In this study, we show that activation of a set of signalling molecules, including MAPK, phosphatidylinositol-3-OH kinase (PI(3)K) and Src, is required for Neu/ErbB2-dependent lamellipodia formation and for motility of breast carcinoma cells. Stimulation of these molecules, however, failed to induce efficient cell migration in the absence of Neu/ErbB2 phosphorylation at Tyr 1201 or Tyr 1227. We describe a novel molecule, Memo (mediator of ErbB2-driven cell motility), that interacts with a phospho-Tyr 1227-containing peptide, most probably through the Shc adaptor protein. After Neu/ErbB2 activation, Memo-defective cells form actin fibres and grow lamellipodia, but fail to extend microtubules towards the cell cortex. Our data suggest that Memo controls cell migration by relaying extracellular chemotactic signals to the microtubule cytoskeleton.

A number of cytoplasmic signaling molecules are thought to mediate mitogenic signaling from the activated Neu receptor tyrosine kinase through binding specific phosphotyrosine residues located within the intracellular portion of Neu/c-ErbB-2. An activated neu oncogene containing tyrosine-to-phenylalanine substitutions at each of the known autophosphorylation sites was generated and assessed for its specific transforming potential in Rat1 and NIH 3T3 fibroblasts. Mutation of these sites resulted in a dramatic impairment of the transforming potential of neu. To assess the role of these tyrosine phosphorylation sites in cellular transformation, the transforming potential of a series of mutants in which individual tyrosine residues were restored to this transformation-debilitated neu mutant was evaluated. Reversion of any one of four mutated sites to tyrosine residues restored wild-type transforming activity. While each of these transforming mutants displayed Rasdependent signaling, the transforming activity of two of these mutants was correlated with their ability to bind either the GRB2 or SHC adapter molecules that couple receptor tyrosine kinases to the Ras signaling pathway. By contrast, restoration of a tyrosine residue located at position 1028 completely suppressed the basal transforming activity of this mutated neu molecule or other transforming neu molecules which possessed single tyrosine residues. These data argue that the transforming potential of activated neu is mediated both by positive and negative regulatory tyrosine phosphorylation sites.The neu/c-ErbB-2 gene encodes a 185-kDa transmembrane receptor tyrosine kinase that is a member of the epidermal growth factor receptor (EGFR) family (4,15,76). Elevated expression of the EGFR family members has been implicated in the etiology of human ovarian and breast cancers (reviewed in references 31 and 41). In particular, amplification and consequent overexpression of the neu receptor tyrosine kinase have been observed in a large proportion of primary human breast cancers (58,64,65). Consistent with these observations, expression of either wild-type neu or a constitutively active mutant (V664E, neuNT [3,5]) in the mammary epithelia of transgenic mice results in the induction of metastatic mammary tumors (11,27,47). The induction of mammary tumors in the wild-type neu transgenic mice correlates with elevated