title: Grb7 upregulation is a molecular adaptation to HER2 signaling inhibition due to removal of Akt-mediated gene repression.
abstract: The efficacy of anti-HER2 therapeutics, such as lapatinib and trastuzumab, is limited by primary and acquired resistance. Cellular adaptations that allow breast cancer cell to survive prolonged HER2 inhibition include de-repression of the transcription factor FOXO3A with consequent estrogen receptor activation, and/or increased HER3 signaling. Here, we used low-density arrays, quantitative PCR, and western blotting to determine how HER2 signaling inhibition with lapatinib or PI3K inhibitors affects the expression of genes involved in breast cancer metastatic spread and overall prognosis. Retroviral transgenesis was used to express constitutively active forms of Akt in the HER2(+) breast cancer cell line SKBR3, and Grb7 in MCF7 cells. Specific gene silencing was obtained by siRNAs transfection. A murine BT474 xenograft cancer model was used to assess the effect of lapatinib on gene expression in vivo. We found that lapatinib induces upregulation of Grb7, an adaptor protein involved in receptor tyrosine kinase signaling and promoting cell survival and cell migration. Grb7 upregulation induced by lapatinib was found to occur in cancer cells in vitro and in vivo. We demonstrate that Grb7 upregulation is recreated by PI3K inhibitors while being prevented by constitutively active Akt. Thus, Grb7 is repressed by PI3K signaling and lapatinib-mediated Akt inhibition is responsible for Grb7 de-repression. Finally, we show that Grb7 removal by RNA-interference reduces breast cancer cell viability and increases the activity of lapatinib. In conclusion, Grb7 upregulation is a potentially adverse consequence of HER2 signaling inhibition. Preventing Grb7 accumulation and/or its interaction with receptor tyrosine kinases may increase the benefit of HER2-targeting drugs.
authors.affiliation: Department of Internal Medicine, Centre of Excellence for Biomedical Research, University of Genoa, Genoa, Italy. A.Nencioni@gmx.net
authors.forename: Alessio
authors.initials: A
authors.lastname: Nencioni
authors.forename: Michele
authors.initials: M
authors.lastname: Cea
authors.forename: Anna
authors.initials: A
authors.lastname: Garuti
authors.forename: Mario
authors.initials: M
authors.lastname: Passalacqua
authors.forename: Lizzia
authors.initials: L
authors.lastname: Raffaghello
authors.forename: Debora
authors.initials: D
authors.lastname: Soncini
authors.forename: Eva
authors.initials: E
authors.lastname: Moran
authors.forename: Gabriele
authors.initials: G
authors.lastname: Zoppoli
authors.forename: Vito
authors.initials: V
authors.lastname: Pistoia
authors.forename: Franco
authors.initials: F
authors.lastname: Patrone
authors.forename: Alberto
authors.initials: A
authors.lastname: Ballestrero
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D051382:GRB7 Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D000077341:Lapatinib; D008325:Mammary Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D011799:Quinazolines / Q000494:pharmacology; D034622:RNA Interference; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014183:Transplantation, Heterologous; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D051382:GRB7 Adaptor Protein; D000077341:Lapatinib; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011799:Quinazolines; D018719:Erb-b2 Receptor Tyrosine Kinases