Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

Resistance to Herceptin represents a significant challenge for successful treatment of HER2-positive breast cancer. Here, we show that in Herceptin-sensitive cells, FOXO3a regulates specific miRNAs to control IGF2 and IRS1 expression, retaining basic IGF2/IGF-1R/IRS1 signaling. The basic activity maintains expression of PPP3CB, a subunit of the serine/threonine-protein phosphatase 2B, to restrict FOXO3a phosphorylation (p-FOXO3a), inducing IGF2- and IRS1-targeting miRNAs. However, in Herceptin-resistant cells, p-FOXO3a levels are elevated due to transcriptional suppression of PPP3CB, disrupting the negative feedback inhibition loop formed by FOXO3a and the miRNAs, thereby upregulating IGF2 and IRS1. Moreover, we detect significantly increased IGF2 in blood and IRS1 in the tumors of breast cancer patients with poor response to Herceptin-containing regimens. Collectively, we demonstrate that the IGF2/IGF-1R/IRS1 signaling is aberrantly activated in Herceptin-resistant breast cancer via disruption of the FOXO3a-miRNA negative feedback inhibition. Such insights provide avenues to identify predictive biomarkers and effective strategies overcoming Herceptin resistance.

Signaling Pathway of GP88 (Progranulin) in Breast Cancer Cells: Upregulation and Phosphorylation of c-myc by GP88/Progranulin in Her2-Overexpressing Breast Cancer Cells

Her2 is a receptor tyrosine kinase overexpressed in 25% of breast tumors. We have shown that the 88 kDa autocrine growth and survival factor GP88 (progranulin) stimulated Her2 phosphorylation and proliferation and conferred Herceptin resistance in Her2-overexpressing cells. Herein, we report that GP88 stimulates c-myc phosphorylation and upregulates c-myc levels in Her2-overexpressing cells. c-myc phosphorylation and upregulation by GP88 were not observed in non-Her2-overexpressing breast cancer cells. c-myc activation was inhibited upon treatment with ERK, PI3 kinase, and c-src pathway inhibitors, U0126, LY294002, and PP2. GP88 also stimulated c-src phosphorylation, a known upstream regulator of c-myc. Thus, we describe here a signaling pathway for GP88 in Her2-overexpressing cells, with GP88 stimulating Src phosphorylation, followed by phosphorylation and upregulation of c-myc. These data would suggest that targeting GP88 could provide a novel treatment approach in breast cancer.

The basal phenotype as a clinically relevant indicator of trastuzumab resistance in HER2+ breast cancer.

154 Background: Trastuzumab (Herceptin) resistance remains a clinical challenge but the mechanism is not well understood. Recently, studies have identified a subset of Her2+ breast cancer called basal HER2 that expresses basal genes. We investigated the effect of basal gene expression on Herceptin response in HER2+ breast cancer cell lines and on prognosis in HER2+ breast cancer patients. Methods: Non-basal (BT474, SKBR3) and basal (HCC1569, HCC1954, JIMT-1) HER2+ cell lines were chosen based on basal cytokeratin expression. Cell proliferation was assessed after treatment with vehicle, Herceptin (H), Paclitaxel (P), H+P, Akt Inhibitor (AI), and H+AI. HER2 signaling was examined using immunoblotting of p-Akt and p-ERK. Because breast cancer stem cells (BCSC) are linked to basal breast tumors and treatment resistance, we assessed BCSC activity using mammosphere formation and aldehyde dehydrogenase (ALDH) positivity. Immunohistochemical staining of HER2+ breast cancers for basal markers CK5/6, CK14, and EGFR was correlated with clinicopathologic features and survival in 88 patients with Stage 1-3 HER2+ breast cancer treated with Herceptin. Results: Basal HER2 cells were resistant to Herceptin compared to non-basal HER2 cells but this resistance was overcome by Akt inhibition. Immunoblotting showed that non-basal HER2 cells had decreased p-Akt after Herceptin treatment which was not seen in basal HER2 cells. There was no difference in p-ERK levels after Herceptin therapy in all cell lines. Basal HER2 cells had increased mammosphere formation and ALDH positivity suggesting higher stem cell activity compared to non-basal HER2 cells. Of the HER2+ patients, 33/88 (37.5%) expressed at least one basal marker. Basal Her2 tumors were associated with higher grade (p = 0.04) and more ER/PR negativity (p < 0.01). CK14 expression correlated with worse overall survival by log-rank test (p = 0.02), while EGFR showed a similar trend (p = 0.06). Conclusions: Basal HER2 breast cancer cell lines have Herceptin resistance which may be due to constitutively active Akt signaling and increased stem cell activity. Clinically, basal marker expression predicts Herceptin resistance and worse outcomes in HER2+ breast cancer patients.