Prognostic value of Maspin protein level in patients with triple negative breast cancer.

The search for prognostic markers in breast cancer has bumped into a typical feature of these tumors, intra and intertumoral heterogeneity. Changes in the expression profile, localization of these proteins or shedding to the surrounding stroma can be useful in the search for new markers. In this context, classification by molecular subtypes can bring perspectives for both diagnosis and screening for appropriate treatments. However, the Triple Negative (TN) subtype, which is already the one with the worst prognosis, lacks appropriate and consistent molecular markers. In this work, we analyzed 346 human breast cancer samples in tissue microarrays (TMA) from cases diagnosed with invasive breast carcinoma to assess the expression and localization pattern of Maspin and their correlation with clinical parameters. To complement our findings, we also used TCGA data to analyze the mRNA levels of these respective genes. Our data suggests that the TN subtype demonstrates a higher level of cytoplasmic Maspin compared to the other subtypes. Maspin transcript levels follow the same trend. However, TN patients with lower Maspin expression tend to have worse overall survival and free-survival metastasis rates. Finally, we used Maspin expression data to verify possible relationships with the clinicopathological information of our cohort. Our univariate analyses indicate that Maspin is related to the expression of estrogen receptor (ER) and progesterone receptor (PR). Furthermore, Maspin expression levels also showed correlation with Scarff-Bloom-Richardson (SBR) parameter, and stromal Maspin showed a relationship with lymph node involvement. Our data is not consistently robust enough to categorize Maspin as a prognostic marker. However, it does indicate a change in the expression profile within the TN subtype.

PI3K-AKT, JAK2-STAT3 pathways and cell-cell contact regulate maspin subcellular localization.

BACKGROUND: Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies indicate that nuclear localization is essential for maspin tumor suppression activity. We have previously shown that the EGFR activation leads to maspin nuclear localization in MCF-10A cells. The present study investigated which EGFR downstream signaling molecules are involved in maspin nuclear localization and explored a possible role of cell-cell contact in this process. METHODS: MCF-10A cells were treated with pharmacological inhibitors against EGFR downstream pathways followed by EGF treatment. Maspin subcellular localization was determined by immunofluorescence. Proteomic and interactome analyses were conducted to identify maspin-binding proteins in EGF-treated cells only. To investigate the role of cell-cell contact these cells were either treated with chelating agents or plated on different cell densities. Maspin and E-cadherin subcellular localization was determined by immunofluorescence. RESULTS: We found that PI3K-Akt and JAK2-STAT3, but not MAP kinase pathway, regulate EGF-induced maspin nuclear accumulation in MCF-10A cells. We observed that maspin is predominantly nuclear in sparse cell culture, but it is redistributed to the cytoplasm in confluent cells even in the presence of EGF. Proteomic and interactome results suggest a role of maspin on post-transcriptional and translation regulation, protein folding and cell-cell adhesion. CONCLUSIONS: Maspin nuclear accumulation is determined by an interplay between EGFR (via PI3K-Akt and JAK2-STAT3 pathways) and cell-cell contact. Video Abstract.

Identification of a putative nuclear localization signal in the tumor suppressor maspin sheds light on its nuclear import regulation.

The tumor suppressor activity of maspin (mammary serine protease inhibitor) has been associated with its nuclear localization. In this study we explore the regulation of maspin nuclear translocation. An in vitro nuclear import assay suggested that maspin can passively enter the nucleus. However, in silico analysis identified a putative maspin nuclear localization signal (NLS), which was able to mediate the nuclear translocation of a chimeric protein containing this NLS fused to five green fluorescent protein molecules in tandem (5GFP). Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N suppressed this process. Unexpectedly, the full-length maspin fused to 5GFP failed to enter the nucleus. As maspin's putative NLS is partially hidden in its three-dimensional structure, we suggest that maspin nuclear transport could be conformationally regulated. Our results suggest that maspin nuclear translocation involves both passive and active mechanisms.

Tyrosine phosphorylation plays a role in increasing maspin protein levels and its cytoplasmic accumulation.

Maspin is a tumor suppressor with many biological activities, multiple ligands and different subcellular localizations. Its underlying molecular mechanism remains elusive. We hypothesized that phosphorylation might regulate maspin localization and function. Using two-dimensional gel electrophoresis with different focusing power followed by Western blot we identified four different maspin forms with the same molecular weight (42 kDa), but different isoelectric points. Three of these forms were sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated. Sodium peroxidovanadate treatment, a protein-tyrosine phosphatase inhibitor, resulted in a rapid increase in maspin protein levels and cytoplasmic accumulation. These data show that there are three different maspin tyrosine phosphoforms. Inhibition of tyrosine phosphatases increased maspin protein levels and leads to its cytoplasmic accumulation.

The maspin expression in canine mammary tumors: an immunohistochemical and molecular study / A expressão do maspin nos tumores mamários caninos: um estudo imuno-histoquímico e molecular

The serpin maspin, a tumor suppressor in breast cancer was described as an inhibitor of cell migration and inducer of cell adhesion between the basement membrane and extracellular matrix resulting in inhibition of tumor metastasis. In contrast, overexpression of maspin is correlated with poor prognosis in other types of cancer. Little is known about expression, regulation and function of maspin in canine mammary tumors. It was demonstrated in this study, a loss of maspin expression in malignant canine mammary cells compared with a pool of normal canine mammary tissue, analyzed by quantitative real-time PCR; weak maspin expression in malignant canine mammary tumors were observed by immunohistochemistry. It was also demonstrated that a correlation with nuclear maspin expression and a good prognosis. It is suggested that maspin could be used as a prognostic marker in canine mammary neoplasia.

O serpin maspin, um supressor tumoral no câncer de mama foi descrito como inibidor de migração celular e indutor de adesão celular entre a membrana basal e a matriz extracelular resultando na inibição da metástase tumoral. Por outro lado, a alta expressão do maspin está relacionada com um mau prognóstico em outros tipos de câncer. Pouco se sabe sobre a expressão, regulação e função do maspin nos tumores mamários caninos. Neste estudo, foi demonstrada uma perda da expressão de maspin nas células mamárias malignas de cães quando comparadas com um pool de tecido mamário normal de cães, analisado por PCR quantitativa em tempo real. Houve uma expressão fraca maspin em preparações de tumores mamários malignos observadas por imuno-histoquímica. Também foi verificado que a expressão nuclear do maspin em tumores mamários caninos está relacionada a um bom prognóstico. Assim, o maspin pode ser utilizado como um marcador prognóstico nas neoplasias mamárias em cães.