Inflammatory Breast Cancer Cell Line SUM149 Research Articles

Leukemia-Associated Primary Granule Proteins (PGPs) Elastase-2 and Proteinase-3 Are Aberrantly Expressed in Solid Tumors: A Potential Therapeutic Target for PR1-Directed Immunotherapy

Neutrophil elastase (ELA2) and proteinase-3 (PRTN3) are the source proteins for the nonomeric HLA-A2-restricted peptide PR1 (VLQELNVTV), a recognized leukemia-associated antigen (LAA). In myeloid leukemia, high expression of ELA2 and PRTN3 has been correlated with improved clinical outcomes following hematopoietic stem cell transplant (HSCT). Vaccination with PR-1 peptide showed efficacy in myeloid leukemia and myelodysplastic syndrome, and was associated with immunologic response (≥ 2 fold increase in PR1-specific cytotoxic T lymphocytes (PR1-CTL)) and long-lasting clinical remissions. PR1-CTLwere shown to mediate immunity elicited by PR-1 vaccine, as they preferentially recognize and kill myeloid leukemia cells. Since ELA2 and PRTN3 have been reported in breast cancer biopsies, we hypothesized that ELA2 and PRTN3 may be mislocalized in solid tumors, leading to MHC-I presentation, therefore providing a therapeutic target for PR1 vaccine. To study the subcellular localization of ELA2 and PRTN3, the cytoplasmic, nuclear, membrane, and golgi/ER fractions were obtained from inflammatory breast cancer (IBC) (MDA-IBC1 and SUM149) and melanoma (526, 624, 888 and 926) cell lines. ELA2 was preferentially expressed in the cytoplasmic fraction in the IBC cell line SUM149, while PRTN3 was expressed in the membrane and cytoskeletal fractions of the IBC cell line MDA-IBC1. In the melanoma cell lines, ELA2 was expressed in the cytoplasmic fractions, while PRTN3 was noted in the nuclear, cytoplasmic and cytoskeletal fractions. Furthermore, the HLA-A2+ melanoma cell line 888 was susceptible to lysis by PR1-CTL (approximately 30% lysis; Effector:Target ratio=1:2). Together, these results demonstrate that ELA2 and PRTN3 are aberrantly localized in non-hematopoietic cell lines, and that this can be associated with susceptibility to PR1- CTL lysis. In particular, PR1 is a potential immunotherapeutic target for patients with melanoma and breast cancer.

WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer

BackgroundInflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer. We found concordant and consistent alterations of two genes in 90% of IBC tumors when compared with stage-matched non-IBC tumors: overexpression of RhoC guanosine triphosphatase and loss of WNT-1 induced secreted protein 3 (WISP3). Further work revealed that RhoC is a transforming oncogene for human mammary epithelial (HME) cells. Despite the aggressiveness of the RhoC-driven phenotype, it does not quantitatively reach that of the true IBC tumors. We have demonstrated that WISP3 has tumor growth and angiogenesis inhibitory functions in IBC. We proposed that RhoC and WISP3 cooperate in the development of IBC.MethodsUsing an antisense approach, we blocked WISP3 expression in HME cells. Cellular proliferation and growth were determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and anchorage-independent growth in a soft agar assay. Vascular endothelial growth factor (VEGF) was measured in conditioned medium by enzyme-linked immunosorbent assay.ResultsAntisense inhibition of WISP3 in HME cells increased RhoC mRNA levels and resulted in an increase in cellular proliferation, anchorage-independent growth and VEGF levels in the conditioned medium. Conversely, restoration of WISP3 expression in the highly malignant IBC cell line SUM149 was able to decrease the expression of RhoC protein.ConclusionWISP3 modulates RhoC expression in HME cells and in the IBC cell line SUM149. This provides further evidence that these two genes act in concert to give rise to the highly aggressive IBC phenotype. We propose a model of this interaction as a starting point for further investigations.