Oncogenic tyrosine kinases have proven to be promising targets for the development of highly effective anticancer drugs. However HER family tyrosine kinase inhibitors (TKIs) show only limited activity against HER2-driven cancers despite effective inhibition of EGFR and HER2 in vivo 1–8. The reasons for this are unclear. Signaling in trans is a key feature of this multimember family and the critically important PI3K/Akt pathway is driven predominantly through transphosphorylation of the kinase-inactive HER3 9,10. We report that HER3 and consequently PI3K/Akt signaling evade inhibition by current HER family TKIs in vitro and in tumors in vivo. This is due to a compensatory shift in HER3 phosphorylation-dephosphorylation equilibrium driven by increased membrane HER3 expression driving the phosphorylation reaction and reduced HER3 phosphatase activity impeding the dephosphorylation reaction. These compensatory changes are driven by Akt mediated negative feedback signaling. Although HER3 is not a direct target of TKIs, HER3 substrate resistance undermines their efficacy and has thus far gone undetected. The experimental abbrogation of HER3 resistance by siRNA knockdown restores potent pro-apoptotic effects to otherwise cytostatic HER TKIs, re-affirming the oncogene-addicted nature of HER2-driven tumors and the therapeutic promise of this oncoprotein target. However, since HER3 signaling is buffered against an incomplete inhibition of HER2 kinase, much more potent TKIs or combination strategies are required to effectively silence oncogenic HER2 signaling. The biologic marker to guide HER TKIs should be the transphosphorylation of HER3.

About 25% of breast cancers harbor the amplified oncogene HER2 and are dependent on HER2 kinase function, identifying HER2 as a vulnerable target for therapy. However,. HER2-HER3 activation is buffered so that it is protected against a nearly two log inhibition of HER2 catalytic activity; this buffering is driven by the negative regulation of HER3 by Akt. We have now further characterized HER2-HER3 signaling activity and shown that the compensatory buffering prevents apoptotic tumor cell death from occurring as a result of the combined loss of MAPK and Akt signaling. To overcome the cancer cells' compensatory mechanisms, we co-administered a PI3K/ mTor inhibitor and a HER2 tyrosinc kinase inhibitor. This treatment strategy proved suboptimal because it induced both tyrosine kinase inhibitor sensitizing and desensitizing effects and robust cross-compensation of MAPK and Akt signaling pathways. Noting that HER2-HER3 activity was completely inhibited by higher, fully inactivating doses of TKI, we then attempted to overcome the cells compensatory buffering with this higher dose. This treatment crippled all downstream signaling and induced tumor apoptosis. Although such high doses of TKI are toxic in vivo when given continuously, we found that intermittent doses of TKI administered to mice produced sequential cycles of tumor apoptosis and ultimately complete tumor regression in mouse models, with much less toxicity. This strategy for inactivation of HER2-HER3 tumorigenic activity is proposed for clinical testing.

The process of cell migration is initiated by protrusion at the leading edge of the cell, the formation of peripheral adhesions, the exertion of force on these adhesions, and finally the release of the adhesions at the rear of the cell. Focal adhesion kinase (FAK) is intimately involved in the regulation of this process, although the precise mechanism(s) whereby FAK regulates cell migration is unclear. We have used two approaches to reduce FAK expression in fibroblasts. Treatment of cells with FAK-specific siRNAs substantially reduced FAK expression and inhibited the spreading of fibroblasts in serum-free conditions, but did not affect the rate of spreading in the presence of serum. In contrast with the wild-type cells, the FAK siRNA-treated cells exhibited multiple extensions during cell spreading. The extensions appeared to be inappropriately formed lamellipodia as evidenced by the localization of cortactin to lamellipodial structures and the inhibition of such structures by expression of dominant-negative Rac. The wild-type phenotype was restored by reexpressing wild-type FAK in the knockdown cells, but not by expression of FAK containing a point mutation at the autophosphorylation site (FAK Y397F). In wound-healing assays, FAK knockdown cells failed to form broad lamellipodia, instead forming multiple leading edges. Similar results were obtained using primary mouse embryo fibroblasts from FAK-flox mice in which Cre-mediated excision was used to ablate the expression of FAK. These data are consistent with a role for FAK in regulating the formation of a leading edge during cell migration by coordinating integrin signaling to direct the correct spatial activation of membrane protrusion.

The human epidermal growth factor receptor (HER) family of transmembrane tyrosine kinases regulate diverse cellular functions in response to extracellular ligands. The deregulation of HER signaling through gene amplification or mutation is seen in many human tumors and an abundance of experimental evidence supports the etiologic role of these events in cancer pathogenesis. In addition, the fact that they are feasible targets for both antibody and small molecule therapeutics has made them highly pursued targets for the development of rationally designed anti-cancer drugs. Several HER-targeting agents have entered clinical practice and this has led to novel discoveries regarding mechanisms of resistance, defining a new generation of challenges for targeted cancer therapies. Here we review recent advances in our understanding of HER signaling and targeting in cancer.

Trask is a recently described transmembrane substrate of Src kinases whose expression and phosphorylation has been correlated with the biology of some cancers. Little is known about the molecular functions of Trask, although its phosphorylation has been associated with cell adhesion. We have studied the effects of Trask phosphorylation on cell adhesion, integrin activation, clustering, and focal adhesion signaling. The small hairpin RNA (shRNA) knockdown of Trask results in increased cell adhesiveness and a failure to properly inactivate focal adhesion signaling, even in the unanchored state. On the contrary, the experimentally induced phosphorylation of Trask results in the inhibition of cell adhesion and inhibition of focal adhesion signaling. This is mediated through the inhibition of integrin clustering without affecting integrin affinity state or ligand binding activity. Furthermore, Trask signaling and focal adhesion signaling inactivate each other and signal in exclusion with each other, constituting a switch that underlies cell anchorage state. These data provide considerable insight into how Trask functions to regulate cell adhesion and reveal a novel pathway through which Src kinases can oppose integrin-mediated cell adhesion.Src family kinases (SFKs) are a family of nonreceptor protein tyrosine kinases with a domain structure consisting of a highly conserved kinase domain as well as an SH2 domain and an SH3 domain, a C-terminal negative-regulatory tyrosine residue, and an N-terminal myristoylation site.

Synopsis HER2-amplified tumors are characterized by constitutive signaling via the HER2-HER3 co-receptor complex. While phosphorylation activity is driven entirely by the HER2 kinase, signal volume generated by the complex is under control of HER3 and a large capacity to increase its signaling output accounts for the resiliency of the HER2-HER3 tumor driver and accounts for the limited efficacies of anti-cancer drugs designed to target it. Here we describe deeper insights into the dynamic nature of HER3 signaling. Signaling output by HER3 is under several modes of regulation including transcriptional, post-transcriptional, translational, post-translational, and localizational control. These redundant mechanisms can each increase HER3 signaling output and are engaged in various degrees depending on how the HER3-PI3K-Akt-mTor signaling network is disturbed. The highly dynamic nature of HER3 expression and signaling, and the plurality of downstream elements and redundant mechanisms that function to ensure HER3 signaling throughput identify HER3 as a major signaling hub in HER2-amplified cancers and a highly resourceful guardian of tumorigenic signaling in these tumors.

NEO-transfected control cells (designated NEO pool) before entering exponential growth in tissue culture. The prolonged growth arrest of RII Cl 37 cells was associated with markedly reduced cyclindependent kinase (CDK)2 activity. Our results demonstrate that p21 induction by autocrine TGF-␤ is responsible for reduced CDK2 activity, which at least partially contributes to prolonged growth arrest and reduced cell proliferation in RII Cl 37 cells. In contrast to RII transfectants, HCT116 cells transfected with chromosome 3 (designated HCT116Ch3), which bears the RII gene, restored the response to exogenous TGF-␤ as well as autocrine TGF-␤ activity. Autocrine TGF-␤ activity in HCT116Ch3 cells induced p21 expression as seen in RII Cl 37 cells; however, in addition to autocrine activity, HCT116Ch3 cells responded to exogenous TGF-␤ as decreased CDK4 expression and reduced pRb phosphorylation mediated a TGF-␤ inhibitory response in these cells. These results indicate that autocrine TGF-␤ regulates the cell cycle through a pathway different from exogenous TGF-␤ in the sense that p21 is a more sensitive effector of the TGF-␤ signaling pathway, which can be induced and saturated by autocrine TGF-␤, whereas CDK4 inhibition is a less sensitive effector, which can only be activated by high levels of exogenous TGF-␤.

The role of the ErbB family in supporting the malignant phenotype was characterized by stable transfection of a single chain antibody (ScFv5R) against ErbB2 containing a KDEL endoplasmic reticulum retention sequence into GEO human colon carcinoma cells. The antibody traps ErbB2 in the endoplasmic reticulum, thereby down-regulating cell surface ErbB2. The transfected cells showed inactivation of ErbB2 tyrosine phosphorylation and reduced heterodimerization of ErbB2 and ErbB3. This resulted in greater sensitivity to apoptosis induced by growth deprivation and delayed tumorigenicity in vivo. Furthermore, decreased heterodimerization of ErbB2 and ErbB3 led to a reorganization in ErbB function in transfected cells as heterodimerization between epidermal growth factor receptor (EGFR) and ErbB3 increased, whereas ErbB3 activation remained almost the same. Importantly, elimination of ErbB2 signaling resulted in an increase in EGFR expression and activation in transfected cells. Increased EGFR activation contributed to the sustained cell survival in transfected cells.Protein-tyrosine kinases are involved in the regulation of cell growth and transformation. The binding of a ligand to the extracellular domain of a receptor tyrosine kinase induces receptor dimerization, activation of the intracellular kinase domain, and autophosphorylation (1). Tyrosine-phosphorylated residues serve as high affinity binding sites for Src homology 2-containing proteins and allow for the modulation of intracellular pathways (2, 3). Constitutive activation of these pathways is apparent in many malignancies and provides maintenance of the malignant phenotype as well as a viable target for cancer therapy.A number of receptor tyrosine kinase subclasses have been described, among which the ErbB family members are of particular interest because of their frequent involvement in human cancer (4, 5). Four members of this family are currently known: the epidermal growth factor (EGF) 1 receptor (EGFR/ ErbB1), ErbB2 (also called Neu/HER2), ErbB3, and ErbB4 (6). All of them share a similar primary structure, but they differ in their ligand specificity and kinase functions (7,8). Increased expression of EGFR is associated with relatively aggressive tumors of the stomach, bladder, lung, and breast (9). An abnormal level of EGFR has been correlated with tumor size and stage in head and neck cancer (10 -12). In this type of cancer, transforming growth factor-␣ and EGFR are both up-regulated (13). In patients with colon carcinoma, increased EGFR mRNA in the tumors is associated with a higher rate of liver metastasis (14). Previous work in this laboratory indicated that inappropriate expression of EGFR and its ligand, transforming growth factor-␣, associates with neoplastic transformation and induces malignant progression of human colon carcinoma (15, 16) as well as activation of ErbB2 (17).Receptor-receptor interactions between ErbB family members were first described by Stern and Kamps (18) and by Wada et al. (19) for EGFR and ErbB2. Ligand-induced receptor...