Natalia Sergina

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 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.

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.