By Kevin E. Noonan --
An important consequence of the biotechnology revolution has been an increased understanding of the molecular biology of cancer. Tyrosine kinases, including the eponymous src oncogene, provide important targets, comprising a family of plasma membrane proteins that in certain incarnations act as growth factor receptors. Until now, therapeutic agents were primarily inhibitors of the tyrosine kinase activity, acting either directly or by preventing dimerization frequently associated with activating the kinase function. However, a report published in Cancer Cell on May 6th illustrated a heretofore unknown activity of these receptors that calls into question the ultimate efficacy of the known inhibitors (see "Survival of Cancer Cells Is Maintained by EGFR Independent of Its Kinase Activity").
The study was performed on the epidermal growth factor receptor, one of a family of such receptors that includes the HER2/neu target of Genentech's breast cancer therapeutic Herceptin®. EGFR (at left) is the target of several anticancer drugs, including several monoclonal antibodies. These include panitumumab, sold by Amgen as Vectibix® for EGFR-expressing metastatic colorectal cancer, and cetuximab, sold by Bristol-Myers Squibb as Erbitux® for EGFR-expressing metastatic colorectal cancer and head and neck cancers. Another class of anti-EGFR drugs inhibit the tyrosine kinase activity; these include gefitinib, sold by AstraZeneca as Iressa® for treating locally advanced or metastatic non-small cell lung cancer (NSCLC), and erlotinib, sold by Genentech as Tarceva® for the treatment of NSCLC and pancreatic cancer. However, the success of these drugs has been more limited than expected, even in light of the otherwise poor prognosis of the typical patients for these therapies.
The authors of the Cancer Cell report propose a reason for the less than expected results: in addition to the known function of EGFR for promoting cellular proliferation associated with the tyrosine kinase activity, EGFR has the newly-found property of promoting active glucose uptake in cancer cells, preventing cell death. The experiments were performed on human cancer cells in vitro, showing that treatment with EGFR inhibitors did not kill the cells (although it did prevent receptor autophosphorylation via the tyrosine kinase activity and reduce cell proliferation). In contrast, transfecting the cells with EGFR-specific siRNA (that reduced EGFR expression) promoted autophagic cell death, i.e. cannibalization of cellular structures and components for energy. Analysis of the cells for intracellular glucose revealed no change in the presence of EGFR inhibitors, but marked reductions after transfection of EGFR siRNA resulting in lowered or abolished EGFR expression. Autophagy could be prevented by increasing the extracellular glucose concentration.
The cancer cells assayed in these experiments expressed two cell surface glucose transporters: GLUT1, which responds to increased extracellular glucose levels and is a passive transporter, and SGLT1, a sodium-dependent ATPase capable of active transport against a glucose concentration gradient. Reduction of EGFR expression had no effect on GLUT1, but reduced SGLT1 detected by Western blot analysis. The autophagic effect was mimicked by treating the cells with SGLT1-specific siRNA. The combination of these results indicated that reduced intracellular glucose concentrations in response to reduced EGFR expression was mediated by SGLT1, and could be compensated by increased GLUT1-mediated glucose transport in response to increased extracellular glucose concentrations. RT-PCR experiments on SGLT1 mRNA showed that SGLT1 gene expression was unaffected by EGFR downregulation, and treatment of proteosome inhibitors prevented reduction of cellular glucose levels mediated by SGLT1, establishing that SGLT1 degradation was the mechanism responsible for the observed effects. Physical interaction between EGFR and SGLT1 was shown by co-immunoprecipitation using anti-EGFR antibodies, and this interaction was shown in MCF-7 cells (which express only very low levels of EGFR) transfected with wildtype as well as tyrosine kinase mutant EGFR. The specificity of these effects was demonstrated by inhibiting endogenous EGFR expression using a 5' untranslated region (UTR)-specific siRNA, and rescuing the phenotype by transfecting the cells with wildtype or tyrosine kinase mutant EGFR species lacking the 5' UTR. Finally, the specificity of SGLT1 involvement in autophagy caused by reduced intracellular glucose concentrations was established using MCF-7 cells (which do not express SGLT1) showing marked sensitivity to reductions in extracellular glucose in the culture media.
These results provide an explanation for the persistence of cancer cells in the face of EGFR-targeted therapies, but also suggest a molecular explanation for the relative failure of these agents to be more effective as anticancer agents. Importantly, these results also suggest that combination therapies, perhaps with anti-metabolic or antiglycolytic agents, may provide improvements in treatment for EGFR-expressing tumors.
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