David Dankort

BrafV600E cooperates with Pten loss to induce metastatic melanoma

Mutational activation of BRAF is the earliest and most common genetic alteration in human melanoma. 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. Notably, combined treatment with rapamycin and PD325901 led to shrinkage of established melanomas. These mice, engineered with a common genetic profile to human melanoma, provide a system to study melanomas cardinal feature of metastasis and for preclinical evaluation of agents designed to prevent or treat metastatic disease.

Mammary tumors expressing the neu proto-oncogene possess elevated c-Src tyrosine kinase activity.

Amplification and overexpression of the neu (c-erbB2) proto-oncogene has been implicated in the pathogenesis of 20 to 30% of human breast cancers. Although the activation of Neu receptor tyrosine kinase appears to be a pivotal step during mammary tumorigenesis, the mechanism by which Neu signals cell proliferation is unclear. Molecules bearing a domain shared by the c-Src proto-oncogene (Src homology 2) are thought to be involved in signal transduction from activated receptor tyrosine kinases such as Neu. To test whether c-Src was implicated in Neu-mediated signal transduction, we measured the activity of the c-Src tyrosine kinase in tissue extracts from either mammary tumors or adjacent mammary epithelium derived from transgenic mice expressing a mouse mammary tumor virus promoter/enhancer/unactivated neu fusion gene. The Neu-induced mammary tumors possessed six- to eightfold-higher c-Src kinase activity than the adjacent epithelium. The increase in c-Src tyrosine kinase activity was not due to an increase in the levels of c-Src but rather was a result of the elevation of its specific activity. Moreover, activation of c-Src was correlated with its ability to complex tyrosine-phosphorylated Neu both in vitro and in vivo. Together, these observations suggest that activation of the c-Src tyrosine kinase during mammary tumorigenesis may occur through a direct interaction with activated Neu.

Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors.

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 promoter-enhancer frequently develop mammary carcinomas (L. Bouchard, L. Lamarre, P. J. Tremblay, and P. Jolicoeur, Cell 57:931-936, 1989; C. T. Guy, M. A. Webster, M. Schaller, T. J. Parson, R. D. Cardiff, and W. J. Muller, Proc. Natl. Acad. Sci. USA 89:10578-10582, 1992; W. J. Muller, E. Sinn, R. Wallace, P. K. Pattengale, and P. 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 receptors 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.

Abrogation of BRAFV600E-induced senescence by PI3K pathway activation contributes to melanomagenesis

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 p15(INK4B). 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.

Grb2 and Shc Adapter Proteins Play Distinct Roles in Neu (ErbB-2)-Induced Mammary Tumorigenesis: Implications for Human Breast Cancer

ABSTRACT Amplification of the Neu (ErbB-2 or HER-2) receptor tyrosine kinase occurs in 20 to 30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Y1144 Y1201, Y1227 and Y1253) of the five known Neu autophosphorylation sites can independently mediate transforming signals. The transforming potential of two of these mutants correlates with their capacity to recruit Grb2 directly to Y1144 (YB) or indirectly through Shc to Y1227 (YD). Here, we demonstrate that these transformation-competent neu mutants activate extracellular signal-regulated kinases and stimulate Ets-2-dependent transcription. Although the transforming potential of three of these mutants (YB, YD, and YE) was susceptible to inhibition by Rap1A, a genetic antagonist of Ras, the transforming potential of YC was resistant to inhibition by Rap1A. To further address the significance of these ErbB-2-coupled signaling molecules in induction of mammary cancers, transgenic mice expressing mutant Neu receptors lacking the known autophosphorylation sites (NYPD) or those coupled directly to either Grb2 (YB) or Shc (YD) adapter molecules were derived. In contrast to the NYPD strains, which developed focal mammary tumors after a long latency period with low penetrance, all female mice derived from YB and YD strains rapidly developed mammary tumors. Although female mice from several independent YB or YD lines developed mammary tumors, the YB strains developed lung metastases at substantially higher rates than the YD strains. These observations argue that Grb2 and Shc play important and distinct roles in ErbB-2/Neu-induced mammary tumorigenesis and metastasis.

Distinct Tyrosine Autophosphorylation Sites Negatively and Positively Modulate Neu-Mediated Transformation

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 Ras-dependent 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.

Signal transduction in mammary tumorigenesis: a transgenic perspective.

A number of genes have been implicated in breast cancer development, yet few have been demonstrated to play causative roles in mammary tumor formation. The advent of transgenic mouse and embryonic stem cell technologies now permits manipulation of the mouse genome in such a way as to temporally and spatially control a gene products expression. Thus, the basic researcher now can directly assess the involvement of particular genes in tumorigenesis and disease progression and, in the process, to develop mouse models of human genetic disease. The utility of such technologies is emphasized in transgenic mice expressing genes thought to play important roles in the initiation and progression of mammary carcinomas. As these transgenic strains have been the subject of several reviews, here we focus on two mouse mammary tumor models, Polyomavirus middle T antigen and the Neu/ErbB-2 receptor tyrosine kinase, which are most amenable to study specific signaling pathways in process of mammary tumorigenesis.

Characterization of Melanoma Cells Capable of Propagating Tumors from a Single Cell

Questions persist about the nature and number of cells with tumor-propagating capability in different types of cancer, including melanoma. In part, this is because identification and characterization of purified tumorigenic subsets of cancer cells has not been achieved to date. Here, we report tumor formation after injection of single purified melanoma cells derived from three novel mouse models. Tumor formation occurred after every injection of individual CD34+p75- melanoma cells, with intermediate rates using CD34-p75- cells, and rarely with CD34-p75+ cells. These findings suggest that tumorigenic melanoma cells may be more common than previously thought and establish that multiple distinct populations of melanoma-propagating cells (MPC) can exist within a single tumor. Interestingly, individual CD34-p75- MPCs could regenerate cellular heterogeneity after tumor formation in mice or multiple passages in vitro, whereas CD34+p75- MPCs underwent self-renewal only, showing that reestablishment of tumor heterogeneity is not always a characteristic of individual cells capable of forming tumors. Functionally, single purified MPCs were more resistant to chemotherapy than non-MPCs. We anticipate that purification of these MPCs may allow a more comprehensive evaluation of the molecular features that define tumor-forming capability and chemotherapeutic resistance in melanoma.

Memo mediates ErbB2-driven cell motility

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.

Multiple ErbB-2/Neu Phosphorylation Sites Mediate Transformation through Distinct Effector Proteins.

Amplification of the type I receptor tyrosine kinase ErbB-2 (HER2/Neu) is observed in 20–30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Tyr1144, Tyr1201, Tyr1226/1227, or Tyr1253) of the five known Neu/ErbB-2 autophosphorylation sites can independently mediate transforming signals. The transforming potential of at least two of these autophosphorylation sites (Tyr1144and Tyr1226/1227) has been further correlated with their ability to associate with Grb2 and Shc adapter proteins, respectively. To confirm the specificity of these interactions, we have created a series of second site mutants in these phosphorylation sites. The results showed that Grb2 recruitment to site 1144 is absolutely required for transforming signal from this autophosphorylation site, whereas association of Shc-mediated transformation is dependent on conservation of the NPXY motif spanning Tyr1227. A stretch of amino acid identity around tyrosines 1201 (ENPEYLTP)and 1253 (ENPEYLDL) exists, and mutation of key residues within this motif reveals distinct requirements for an intact protein tyrosine-binding protein (NPXY). We show that DOK-R, a protein tyrosine-binding site-containing protein implicated in Ras signaling, interacts with Neu/ErbB-2 at Tyr1253 as do two unidentified proteins, p150 and p34, the latter correlating with transformation. Together these data argue that ErbB-2/Neu is capable of mediating transformation through distinct effector pathways.