Abstract P3-06-15: Notch3 as a predictor of GSI sensitivity in distinct subtypes of triple negative breast cancer
Authors: Shah D , Osipo C 
Published: May 2016

Background: Breast cancer is the second leading cause of cancer-associated death in women. Triple negative breast cancer (TNBC) accounts for 10-15% of breast cancer, lacks expression of ER, PR, and HER2 gene amplification. Due to lack of targeted therapy, TNBC has the worst prognosis. TNBC is a heterogeneous disease with six distinct subtypes. Current treatment includes combination of surgery, radiation therapy and chemotherapy. Notch signaling is increased in TNBC and predicts for worst overall outcome. The goal of the study is to identify novel Notch-biomarkers that predict sensitivity of subtypes of TNBC to Notch inhibition in both bulk and cancer stem cells.

Methods: Two subtypes of TNBC, Mesenchymal stem-like MDA-MB-231 and basal like MDA-MB-468 cells were used. Notch signaling was evaluated using both RNA-sequencing and quantitative real time PCR (q-RTPCR). Cancer stem cell markers were evaluated using Aldefluor assay, CD44 high/ CD24 low expression, and mammosphere forming assay. To study the effect of chemotherapy and Notch, MDA-MB-231 and MDA-MB-468 cells were treated with carboplatin or a pan Notch inhibitor- gamma secretase inhibitor (GSI) or combination of both and cell viability was measured using the trypan blue exclusion test.

Results: The RNA-seq and RT-PCR results showed that Notch3 is differentially expressed in the two cell lines. Notch3 was knocked down using siRNA and its effect on cell viability was assessed after GSI or carboplatin or combination treatment. MDA-MB-468 cells had higher Notch receptors and activity, most notably Notch3. MDA-MB-468 cells had high levels of Aldefluor whereas MDA-MB-231 cells showed higher levels of CD44 High/CD24 Low expression. MDA-MB-468 cells had higher mammosphere forming efficiency (MFE) compared to MDA-MB-231. Notch inhibition decreased MFE of MDA-MB-468 whereas it increased MFE of MDA-MB-231 cells. MDA-MB-468 cells have lower IC50 for carboplatin or GSI compared to MDA-MB-231 cells, as measured by cell viability assay. However, combination treatment lowered IC50 for GSI in MDA-MB-231 cells as compared to MDA-MB-468 cells. Since Notch3 levels were strikingly different between the two cell lines, we hypothesized that Notch3 levels are necessary for GSI sensitivity. Carboplatin treatment increased Notch3 in MDA-MB-231 cells and the increase in Notch3 could be the reason for lower IC50 of GSI during combination treatment as increased Notch3 would provide the substrate for gamma-secretase. In MDA-MB-468 cells, Notch3 knockdown significantly reduced cell viability and was similar to GSI, suggesting that GSI acts through Notch3. Furthermore, Notch3 knockdown and carboplatin treatment reduced viability comparable to carboplatin and GSI treatment, reinforcing that the GSI acts through Notch3 in TNBC. Using the Kaplan-Meier Plotter, high Notch3 predicted poor recurrence free survival post chemotherapy in patients with ER-, HER2-, basal breast cancer.

Conclusions: GSI acts through Notch3 in two TNBC subtypes and combination of chemotherapy with Notch inhibition results in a better outcome as compared to either drug alone. Future experiments would elucidate the role of Notch3 inhibition in targeting cancer stem cells post chemotherapy treatment in different subtypes of TNBC.

Citation Format: Shah D, Osipo C. Notch3 as a predictor of GSI sensitivity in distinct subtypes of triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-15.