SUMMARY Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, whereas prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

Risk factors for the newly identified "intrinsic" breast cancer subtypes (luminal A, luminal B, basal-like and human epidermal growth factor receptor 2-positive/estrogen receptor-negative) were determined in the Carolina Breast Cancer Study, a population-based, case-control study of African-American and white women. Immunohistochemical markers were used to subtype 1,424 cases of invasive and in situ breast cancer, and case subtypes were compared to 2,022 controls. Luminal A, the most common subtype, exhibited risk factors typically reported for breast cancer in previous studies, including inverse associations for increased parity and younger age at first full-term pregnancy. Basal-like cases exhibited several associations that were opposite to those observed for luminal A, including increased risk for parity and younger age at first term full-term pregnancy. Longer duration breastfeeding, increasing number of children breastfed, and increasing number of months breastfeeding per child were each associated with reduced risk of basal-like breast cancer, but not luminal A. Women with multiple live births who did not breastfeed and women who used medications to suppress lactation were at increased risk of basal-like, but not luminal A, breast cancer. Elevated waist-hip ratio was associated with increased risk of luminal A in postmenopausal women, and increased risk of basal-like breast cancer in pre- and postmenopausal women. The prevalence of basal-like breast cancer was highest among premenopausal African-American women, who also showed the highest prevalence of basal-like risk factors. Among younger African-American women, we estimate that up to 68% of basal-like breast cancer could be prevented by promoting breastfeeding and reducing abdominal adiposity.

Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. (12). There are several mechanisms by which growth factor receptors can be rendered transforming. Retroviral transduction of protooncogenes can result in truncation and mutation of the normal version of the gene. This has been shown for the epidermal growth factor receptor (EGFR) (v-erbB) (18), colony-stimulating factor 1 receptor (CSF-1R) (v-fms) (68), and ros (v-ros) (52) genes. Overexpression of an otherwise normal receptor, with or without the concomitant application of ligand, can also result in neoplastic transformation, as shown for the insulinlike growth factor 1 receptor (38), EGFR (15, 77), CSF-1R (65), eph (51), and neu (16) HOS (10), has been demonstrated to be the cell surface receptor for hepatocyte growth factor, a potential growth factor for a broad spectrum of cell types as well as a mediator of liver regeneration (7). In addition, CSF-1R mediates the pleiotropic effects of its cognate ligand, CSF-1. Together, these two molecules stimulate the proliferation and differentiation of cells of the macrophage lineage (68).In an effort to determine genes involved in the progression of chronic myelogenous leukemia (CML) to acute-phase leukemia, we previously reported the identification of a transforming gene in the DNAs of two patients with CML (47). Molecular cloning and characterization indicate that this gene, which we term axl (from the Greek word anexelekto, or uncontrolled), is a receptor tyrosine kinase with a structure novel among tyrosine kinases. Our data indicate that the ax! protein has tyrosine kinase activity and is capable of transforming NIH 3T3 cells. Furthermore, axl's transforming capacity results from overexpression of axl mRNA rather than from structural mutation. MATERIALS AND METHODSIdentification of a transforming gene in CML cells. Transfections and nude mouse tumorigenicity assays were performed as described previously (47). The cell lines AF6295 and AF3642 were derived from secondary nude mouse tumors arising from transfection of DNA from blast crisis and chronic-phase CML patients, respectively. Tumors were isolated from nude mice and digested in the presence of trypsin-EDTA. A portion of these cells was then plated in plastic tissue culture flasks.Isolation of cosmid and cDNA clones. DNA fragments for cosmid cloning were generated by partial MboI digests of genomic DNA from a secondary nude mouse tumor cell line, AF6295. The restricted DNA fragment...

Aberrant activation of tyrosine kinases is linked causally to human cancers. Activated Cdc42-associated kinase (Ack1), an intracellular tyrosine kinase, has primarily been studied for its signaling properties but has not been linked to specific pathologic conditions. Herein, we report that expression of activated Ack1 in LNCaP cells, while minimally increasing growth in culture, enhanced anchorage-independent growth in vitro and dramatically accelerated tumorigenesis in nude mice. Molecular chaperone heat shock protein 90B (Hsp90B)-bound Ack1 and treatment of cells with geldanamycin, a Hsp90 inhibitor, inhibited Ack1 kinase activity and suppressed tumorigenesis. Further, we identify the tumor suppressor WW domain containing oxidoreductase (Wwox) as an Ack1-interacting protein. Activated Ack1 tyrosine phosphorylated Wwox, leading to rapid dissociation of the Ack1-Wwox complex and concomitant Wwox polyubiquitination followed by degradation. Tyrosine phosphorylation of Wwox was critical for its degradation, as splice variant Wwox#5-8 that was not phosphorylated by Ack1 failed to undergo polyubiquitination and degradation. It has been reported that phosphorylation of Wwox at Tyr 33 stimulated its proapoptotic activity. We observed that Y33F Wwox mutant was still tyrosine phosphorylated and polyubiquitinated by Ack1 action. Site-directed mutagenesis revealed that activated Ack1 primarily phosphorylated Wwox at Tyr 287 , suggesting that phosphorylation of distinct tyrosine residues activate or degrade Wwox. Primary androgen-independent prostate tumors but not benign prostate showed increased tyrosinephosphorylated Ack1 and decreased Wwox. Taken together, these data indicate that Ack1 stimulated prostate tumorigenesis in part by negatively regulating the proapoptotic tumor suppressor, Wwox. Further, these findings suggest that Ack1 could be a novel therapeutic target for prostate cancer. (Cancer Res 2005; 65(22): 10514-23)

Many G protein-coupled receptors (e.g. that of angiotensin II) activate phospholipase C␤, initially increasing intracellular calcium and activating protein kinase C. In the WB and GN4 rat liver epithelial cell lines, agonistinduced calcium signals also stimulate tyrosine phosphorylation and subsequently increase the activity of c-Jun N-terminal kinase (JNK). We have now purified the major calcium-dependent tyrosine kinase (CADTK), and by peptide and nucleic acid sequencing identified it as a rat homologue of human PYK2. CADTK/PYK2 is most closely related to p125 FAK and both enzymes are expressed in WB and GN4 cells. Angiotensin II, which only slightly increases p125 FAK tyrosine phosphorylation in GN4 cells, substantially increased CADTK tyrosine autophosphorylation and kinase activity. Agonists for other G protein-coupled receptors (e.g. LPA), or those increasing intracellular calcium (thapsigargin), also stimulated CADTK. In comparing the two rat liver cell lines, GN4 cells exhibited ϳ 5-fold greater angiotensin II-and thapsigargin-dependent CADTK activation thanWBcells.AlthoughmaximalJNKactivationbystressdependent pathways (e.g. UV and anisomycin) was equivalent in the two cell lines, calcium-dependent JNK activation was 5-fold greater in GN4, correlating with CADTK activation. In contrast to JNK, the thapsigargin-dependent calcium signal did not activate mitogen-activated protein kinase and Ang II-dependent mitogen-activated protein kinase activation was not correlated with CADTK activation. Finally, while some stress-dependent activators of the JNK pathway (NaCl and sorbitol) stimulated CADTK, others (anisomycin, UV, and TNF␣) did not. In summary, cells expressing CADTK/PYK2 appear to have two alternative JNK activation pathways: one stressactivated and the other calcium-dependent.