HER3 signalling is regulated through a multitude of redundant mechanisms in HER2-driven tumour cells.

HER2 (human epidermal growth factor receptor-2)-amplified tumours are characterized by constitutive signalling via the HER2-HER3 co-receptor complex. Although phosphorylation activity is driven entirely by the HER2 kinase, signal volume generated by the complex is under the control of HER3, and a large capacity to increase its signalling output accounts for the resiliency of the HER2-HER3 tumour driver and accounts for the limited efficacies of anti-cancer drugs designed to target it. In the present paper we describe deeper insights into the dynamic nature of HER3 signalling. Signalling 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 signalling output and are engaged in various degrees depending on how the HER3/PI3K (phosphoinositide 3-kinase)/Akt/mTOR (mammalian target of rapamycin) signalling network is disturbed. The highly dynamic nature of HER3 expression and signalling, and the plurality of downstream elements and redundant mechanisms that function to ensure HER3 signalling throughput identify HER3 as a major signalling hub in HER2-amplified cancers and a highly resourceful guardian of tumorigenic signalling in these tumours.

Phosphorylation of Trask by Src kinases inhibits integrin clustering and functions in exclusion with focal adhesion signaling.

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.

Resiliency and vulnerability in the HER2-HER3 tumorigenic driver.

About 25% of breast cancers harbor the amplified oncogene human epidermal growth factor receptor 2 (HER2) and are dependent on HER2 kinase function, identifying HER2 as a vulnerable target for therapy. However, HER2-HER3 signaling 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 have shown that the compensatory buffering prevents apoptotic tumor cell death from occurring as a result of the combined loss of mitogen-activated protein kinase (MAPK) and Akt signaling. To overcome the cancer cells' compensatory mechanisms, we coadministered a phosphoinositide 3-kinase-mammalian target of rapamycin inhibitor and a HER2 tyrosine kinase inhibitor (TKI). This treatment strategy proved equivocal because it induced both TKI-sensitizing and TKI-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 little toxicity. This strategy for inactivation of HER2-HER3 tumorigenic activity is proposed for clinical testing.

The HER family and cancer: emerging molecular mechanisms and therapeutic targets.

The human epidermal growth factor receptor (HER) family of transmembrane tyrosine kinases regulates 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 etiological 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 anticancer drugs. Several HER-targeting agents have entered clinical practice and this has led to novel discoveries regarding the mechanisms of resistance, which has defined a new generation of challenges for targeted cancer therapies. Here, we review recent advances in our understanding of HER signaling and targeting in cancer.

Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3.

Oncogenic tyrosine kinases have proved to be promising targets for the development of highly effective anticancer drugs. However, tyrosine kinase inhibitors (TKIs) against the human epidermal growth factor receptor (HER) family show only limited activity against HER2-driven breast cancers, despite effective inhibition of epidermal growth factor receptor (EGFR) and HER2 in vivo. The reasons for this are unclear. Signalling in trans is a key feature of this multimember family and the critically important phosphatidylinositol-3-OH kinase (PI(3)K)/Akt pathway is driven predominantly through transphosphorylation of the kinase-inactive HER3 (refs 9, 10). Here we show that HER3 and consequently PI(3)K/Akt signalling evade inhibition by current HER-family TKIs in vitro and in tumours in vivo. This is due to a compensatory shift in the HER3 phosphorylation-dephosphorylation equilibrium, driven by increased membrane HER3 expression driving the phosphorylation reaction and by reduced HER3 phosphatase activity impeding the dephosphorylation reaction. These compensatory changes are driven by Akt-mediated negative-feedback signalling. Although HER3 is not a direct target of TKIs, HER3 substrate resistance undermines their efficacy and has thus far gone undetected. The experimental abrogation of HER3 resistance by small interfering RNA knockdown restores potent pro-apoptotic activity to otherwise cytostatic HER TKIs, re-affirming the oncogene-addicted nature of HER2-driven tumours and the therapeutic promise of this oncoprotein target. However, because HER3 signalling is buffered against an incomplete inhibition of HER2 kinase, much more potent TKIs or combination strategies are required to silence oncogenic HER2 signalling effectively. The biologic marker with which to assess the efficacy of HER TKIs should be the transphosphorylation of HER3 rather than autophosphorylation.

Focal adhesion kinase is required for the spatial organization of the leading edge in migrating cells.

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.

Reorganization of ErbB family and cell survival signaling after Knock-down of ErbB2 in colon cancer cells.

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.

Autocrine and exogenous transforming growth factor beta control cell cycle inhibition through pathways with different sensitivity.

Human colon carcinoma cells HCT116 that lack transforming growth factor beta (TGF-beta) type II receptor (RII) demonstrated restoration of autocrine TGF-beta activity upon reexpression of RII without restoring inhibitory responses to exogenous TGF-beta treatment. RII transfectants (designated RII Cl 37) had a longer lag phase relative to 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 cyclin-dependent kinase (CDK)2 activity. Our results demonstrate that p21 induction by autocrine TGF-beta 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-beta as well as autocrine TGF-beta activity. Autocrine TGF-beta 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-beta as decreased CDK4 expression and reduced pRb phosphorylation mediated a TGF-beta inhibitory response in these cells. These results indicate that autocrine TGF-beta regulates the cell cycle through a pathway different from exogenous TGF-beta in the sense that p21 is a more sensitive effector of the TGF-beta signaling pathway, which can be induced and saturated by autocrine TGF-beta, whereas CDK4 inhibition is a less sensitive effector, which can only be activated by high levels of exogenous TGF-beta

The role of transforming growth factor alpha in determining growth factor independence.

Growth factor independence is a hallmark of malignancy that is attributed to the development of autocrine growth factor loops in cancer cells. However, growth factor-dependent normal cells also exhibit autocrine activity, thus raising the issue of how endogenously produced activity in cancer cells differs in a manner that leads to growth factor independence. We have examined this issue by comparing growth factor-independent HCT116 human colon carcinoma cells with a growth factor-dependent subcompartment of malignant cells designated HCT116b that was isolated from the same patient tumor. Therefore, the development of the growth factor-independent phenotype represents clonal progression within the tumor in vivo. The growth factor independence of HCT116 cells was shown to be dependent on autocrine transforming growth factor (TGF)-alpha activity, yet the isoparental HCT116b subcompartment showed similar levels of TGF-alpha expression as HCT116 when cells were in exponential growth. When both cell lines were growth arrested by nutrient deprivation, HCT116b cells required nutrient replenishment and growth factors for reinitiation of DNA synthesis, whereas HCT116 cells required only nutrient replenishment. In contrast to growth factor-dependent HCT116b cells, the HCT116 cells showed up-regulation of TGF-alpha expression during growth arrest as a result of enhanced transcription. This increased TGF-alpha expression in quiescent HCT116 cells was associated with constitutive epidermal growth factor receptor (EGFR) activation in the growth-arrested state, whereas growth-arrested HCT116b cells did not show EGFR activation. TGF-alpha antisense transfection of HCT116 cells showed that EGFR activation was due to increased TGF-alpha expression. Pretreatment of growth-arrested HCT116 cells with AG1478, a selective inhibitor of EGFR tyrosine kinase activity, blocked the reinitiation of DNA synthesis, demonstrating that growth factor independence was due to the increased TGF-alpha expression and EGFR activation of these cells in growth arrest relative to growth factor-dependent HCT116b cells. Importantly, the level of EGFR activation in growth-arrested HCT116 cells was only slightly higher than that of exponential cells, indicating that it was inappropriate EGFR activation in growth arrest rather than the amplitude of activation that generated growth factor independence.