Ability to Generate Patient-Derived Breast Cancer Xenografts Is Enhanced in Chemoresistant Disease and Predicts Poor Patient Outcomes.

BACKGROUND: Breast cancer patients who are resistant to neoadjuvant chemotherapy (NeoCT) have a poor prognosis. There is a pressing need to develop in vivo models of chemo resistant tumors to test novel therapeutics. We hypothesized that patient-derived breast cancer xenografts (BCXs) from chemo- naïve and chemotherapy-exposed tumors can provide high fidelity in vivo models for chemoresistant breast cancers. METHODS: Patient tumors and BCXs were characterized with short tandem repeat DNA fingerprinting, reverse phase protein arrays, molecular inversion probe arrays, and next generation sequencing. RESULTS: Forty-eight breast cancers (24 post-chemotherapy, 24 chemo-naïve) were implanted and 13 BCXs were established (27%). BCX engraftment was higher in TNBC compared to hormone-receptor positive cancer (53.8% vs. 15.6%, p = 0.02), in tumors from patients who received NeoCT (41.7% vs. 8.3%, p = 0.02), and in patients who had progressive disease on NeoCT (85.7% vs. 29.4%, p = 0.02). Twelve patients developed metastases after surgery; in five, BCXs developed before distant relapse. Patients whose tumors developed BCXs had a lower recurrence-free survival (p = 0.015) and overall survival (p<0.001). Genomic losses and gains could be detected in the BCX, and three models demonstrated a transformation to induce mouse tumors. However, overall, somatic mutation profiles including potential drivers were maintained upon implantation and serial passaging. One BCX model was cultured in vitro and re-implanted, maintaining its genomic profile. CONCLUSIONS: BCXs can be established from clinically aggressive breast cancers, especially in TNBC patients with poor response to NeoCT. Future studies will determine the potential of in vivo models for identification of genotype-phenotype correlations and individualization of treatment.

The effect of leucine restriction on Akt/mTOR signaling in breast cancer cell lines in vitro and in vivo.

The mammalian target of rapamycin (mTOR) is a central controller of cell growth and is currently being investigated as a potential target in breast cancer therapy. The essential amino acid leucine has been proposed to regulate mTOR signaling. The objective of this study was to determine whether leucine restriction would inhibit mTOR signaling in breast cancer cells. Leucine restriction did not decrease mTOR signaling in any of the 8 breast cancer cell lines tested. In addition, in vivo administration of a leucine-free diet for up to 4 days did not result in a decrease in phosphorylation of mTOR target proteins in breast cancer xenografts. Further, in 3 different cell lines, an increase in Akt phosphorylation was observed after leucine restriction. This was observed without a decrease in S6K phosphorylation, suggesting a mechanism different from the feedback loop activation of Akt observed with rapamycin treatment. We conclude that leucine restriction is not sufficient to inhibit mTOR signaling in most breast cancer cell lines but is associated with activation of survival molecule Akt, making leucine deprivation an undesirable approach for breast cancer therapy.

Regulation and localization of ribosomal protein S6 kinase 1 isoforms.

Ribosomal protein S6 kinase 1 (S6K1), a critical mediator of cell growth, is overexpressed in breast cancer and is associated with poor prognosis. S6K1 has two known isoforms, p85(S6K1) and p70(S6K1). p85(S6K1) is characterized by 23 additional amino acids in the N-terminus of p70(S6K1). This is thought to target p85(S6K1) to the nucleus, while p70(S6K1) is mainly cytoplasmic. We sought to determine the activation, regulation, and function of p70(S6K1) and p85(S6K1) in breast cancer. We found that most breast cancer cell lines expressed both isoforms. Mitogen-dependent pathways concordantly regulated phosphorylation on T389, S371, and T421/S424. Phosphorylation of both isoforms was inhibited by PI3K/mTOR inhibitors. Mitogen-dependent pathways concordantly regulated the phosphorylation of the two isoforms on T389, S371, and T421/S424. Both isoforms had S6 kinase activity. We also detected a p60 isoform with antibodies to the p70(S6K1) C-terminal but not the N-terminal. Further studies on S6K1 isoforms are warranted for therapeutically targeting this pathway.