NOTCH3 expression in metastatic TNBC cells.

Abstract

e12123 Background: Onset of metastases involves a complex multistep biological process termed the invasion-metastasis cascade, which include dissemination of cancer cells from the primary tumor to secondary organs. NOTCH signaling plays a critical role in promoting Triple Negative Breast Cancer (TNBC) metastasis. Although all four NOTCH receptors show oncogenic properties, the unique role of each of these receptors in the sequential stepwise events that typify the invasion-metastasis cascade remains elusive. Methods: We established unique TNBC cells (TNBC-M14, -M25 and –M40), isolated from patient-derived brain metastasis xenografts (PD-BMXs). NOTCH3 genetic targeting was achieved using Lenti-vector shRNAs. Immunoblot analysis was used to assess NOTCH3 expression. ALDH1 activity was measured using the ALDEFLUOR Kit. To assess self-renewal capacity and resistance to docetaxel-based chemotherapy, TNBC cells were cultured under non-adherent conditions to form mammospheres (MPS). A publicly clinical database ( http://kmplot.com ) was employed to analyze NOTCH3 expression in a selected cohort of 107 lymph- node+ TNBC patients. Results: TNBC-M14, -M25 and -M40 MPS showed NOTCH3 overexpression and higher ALDH1 activity compared to MDA-MB 231 MPS used as control. To investigate the causative role of NOTCH3 in inducing ALDH1 activity, self-renewal capacity and chemoresistance, we infected TNBC MPS with Lenti-vector shRNAs targeting NOTCH3. NOTCH3 geneting targeting induced a significant reduction of ALDH1 activity compared to TNBC MPS infected with scrambled Lenti-shRNAs. NOTCH3 geneting targeting significantly impaired MPS formation and restored sensitivity to docetaxel. The correlation between aberrant NOTCH3 expression and poor outcome in patients with advanced TNBC was validated in a publicly clinical database. Conclusions: These findings provide a compelling preclinical rationale for the design of novel clinical trials that will selectively target NOTCH3 to restore chemosensitivity and to improve the progression-free survival of TNBC patients that lack FDA-approved targeted therapies.