Cyclin E overexpression confers resistance to trastuzumab through noncanonical phosphorylation of SMAD3 in HER2+ breast cancer

Document Type

Article

Publication Date

10-14-2020

Publication Title

Cancer biology & therapy

Abstract

The efficacy of trastuzumab, a treatment for HER2+ breast cancer, can be limited by the development of resistance. Cyclin E (CCNE) overexpression has been implicated in trastuzumab resistance. We sought to uncover a potential mechanism for this trastuzumab resistance and focused on a model of CCNE overexpressing HER2+ breast cancer and noncanonical phosphorylation of the TGF-β signaling protein, SMAD3. Network analysis of transcriptional activity in a HER2+, CCNE overexpressing, trastuzumab-resistant cell line (BT474R2) identified decreased SMAD3 activity was associated with treatment resistance. Immunoblotting showed SMAD3 expression was significantly downregulated in BT474R2 cells (p < .01), and noncanonical phosphorylation of SMAD3 was increased in these CCNE-overexpressing cells. Also, in response to CDK2 inhibition, expression patterns linked to restored canonical SMAD3 signaling, including decreased cMyc and increased cyclin-dependent inhibitor, p15, were identified. The BT474R2 cell line was modified through overexpression of SMAD3 (BT474R2-SMAD3), a mutant construct resistant to CCNE-mediated noncanonical phosphorylation of SMAD3 (BT474R2-5M), and a control (BT474R2-Blank). In vitro studies examining the response to trastuzumab showed increased sensitivity to treatment for BT474R2-5M cells. These findings were then validated in NSG mice inoculated with BT474R2-5M cells or BT474R2 control cells. After treatment with trastuzumab, the NSG mice inoculated with BT474R2-5M cells developed significantly lower tumor volumes (p < .001), when compared to mice inoculated with BT474R2 cells. Taken together, these results indicate that for patients with HER2+ breast cancer, a mechanism of CCNE-mediated trastuzumab resistance, regulated through noncanonical SMAD3 phosphorylation, could be treated with CDK2 inhibition to help enhance the efficacy of trastuzumab therapy.

Medical Subject Headings

Animals; Breast Neoplasms/drug therapy/genetics/pathology; Cell Proliferation; Cyclin E/metabolism; Female; Humans; Mice; Mice; Inbred NOD; Phosphorylation; Smad3 Protein/metabolism; Trastuzumab/pharmacology/therapeutic use; Her2; Smad3; Trastuzumab; breast cancer; cyclin E; fadraciclib

PubMed ID

33054513

ePublication

ePub ahead of print

Volume

21

Issue

11

First Page

994

Last Page

1004

Find It @ Sladen

Share

COinS