PDF file - 201K, HRGalpha, as well as HRGbeta, is capable of stimulating the ability of SKBR3 cells to form colonies and activate components of the mTORC1 signaling pathway. A, SKBR3 cells were seeded in 0.3% agarose-containing complete medium with the addition of 1 nM HRGalpha or 1 nM HRGbeta. Cells were fed every three days with the growth factor-containing medium and colonies were counted on day 13. The experiment was performed in triplicate and the results were averaged and graphed. B, SKBR3 cells were serum-starved for 40-48 h, and were then challenged with HRGalpha for the indicated times. Cell lysates were then collected and analyzed by Western blotting to determine relative levels of phospho-mTOR (S2448), phospho-TSC2 (T1462), phospho-AKT (T308), phospho-AKT (S473), phospho-ribosomal S6 (S235/236), actin, as well as for the total levels of the respective proteins.
ARTICLE ABSTRACT
Overexpression of the receptor tyrosine kinase HER2/ErbB2 (ERBB2) has been linked to a poor prognosis for patients with breast cancer; thus, its activity is a central target for cancer therapy. Likewise, overexpression of heregulin (HRG/NRG1), a growth factor responsible for ErbB2 activation, has also been shown to be a driver of breast cancer progression. Although ErbB2 inhibitors offer a major advancement in the treatment of ErbB2-dependent breast cancers, patients are highly susceptible to developing clinical resistance to these drugs. Therefore, a detailed understanding of the molecular mechanism that underlies HRG/ErbB2-induced tumorigenesis is essential for the development of effective therapeutic strategies for this subset of patients with breast cancer. Here, it was demonstrated that HRG promoted anchorage-independent breast cancer cell growth more potently than EGF, and that the HRG-dependent activation of phosphoinositide 3-kinase and mTORC1 are necessary events for cell transformation. Functional evaluation of two distinct mTOR (MTOR) inhibitors, rapamycin and INK-128, on HRG-dependent signaling activities, uncovered a necessary role for mTORC2 in the regulation of the AKT/TSC2/mTORC1 axis by affecting the phosphorylation of AKT at the PDK1(PDPK1)-dependent site (T308) as well as at the mTORC2-dependent site (S473). The elimination of Rictor (RICTOR), a critical component of mTORC2, is detrimental to both the activation of mTORC1 and HRG-mediated cellular transformation. Similar results were obtained in multiple breast cancer model systems, highlighting an important role for mTORC2 in HRG/ErbB2-dependent breast cancer.Implications: These findings suggest the potential benefits of targeting mTORC2 in HRG/ErbB2-induced breast cancer. Mol Cancer Res; 12(6); 940–52. ©2014 AACR.
