Maspin Subcellular Localization Research Articles

PI3K-AKT, JAK2-STAT3 pathways and cell\u2013cell contact regulate maspin subcellular localization

BackgroundMaspin (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.MethodsMCF-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.ResultsWe 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.ConclusionsMaspin nuclear accumulation is determined by an interplay between EGFR (via PI3K-Akt and JAK2-STAT3 pathways) and cell–cell contact.DeaMyif71VSQHA76EZpJtKVideo

Maspin subcellular expression in wild-type and mutant TP53 gastric cancers.

BACKGROUNDAlthough the role of p53 in the evolution and prognosis of gastric cancer (GC) has been extensively examined, the exact mechanism of action is incompletely understood. In the last years, p53-target genes were supposed to be involved in the p53 pathway. One of them is the tumor-suppressor gene Maspin, which codifies the protein with the same name. Maspin activity depends on its subcellular localization. To our knowledge, the possible role of TP53 gene in Maspin subcellular localization, in GC cells, has not yet been studied in a large number of human samples.AIMTo evaluate the possible role of wild-type and mutated p53 in Maspin subcellular localization.METHODSThe present study included 266 consecutive patients with GC in which TP53 gene status, and mutations in exons 2 to 11, respectively, were analyzed and correlated with immunohistochemical expression of p53 and Maspin.RESULTSNone of the 266 cases showed mutations in exon 9. The rate of TP53 mutations was 33.83%. The mutation rate was slightly higher in distally-located GCs, with a lower degree (≤ 5 buds/ high power fields) of dyscohesivity (P < 0.01). The wild-type cases had a longer survival, compared with mutant GCs, especially in patients without lymph node metastases, despite the high depth of tumor infiltration (P = 0.01). The Dukes-MAC-like staging system was proved to have the most significant independent prognostic value (P < 0.01). The statistical correlations proved that TP53 gene mutations in exon 7 might induce knockdown of Maspin, but wild-type p53 can partially restore nuclear Maspin expression and decrease the metastatic potential of gastric adenocarcinoma cells.CONCLUSIONDownregulated Maspin might be induced by mutations in exon 7 of the TP53 gene but wild-type p53 can partially restore nuclear Maspin expression. These findings should be proved in experimental studies.

Nuclear expression of onco-suppressors nm23-H1 and maspin are associated with lower recurrence rate in laryngeal carcinoma.

The main aim of the study was to preliminarily investigate the possibly related role of nuclear onco-suppressors maspin and nm23-H1, a metastasis suppressor, in laryngeal squamous cell carcinoma (LSCC). Maspin expression pattern and nuclear nm23-H1 expression were ascertained in 62 consecutive LSCCs. Recurrence rate was significantly lower in patients with a nuclear maspin pattern of expression; nuclear nm23-H1 expression was significantly lower in patients who experienced disease recurrence. Disease free survival (DFS) was significantly longer in patients with maspin nuclear pattern or with nuclear nm23-H1 expression ≥10%. A significant association was found between nuclear nm23-H1 expression and maspin pattern of expression in LSCC. KNN discriminant analysis considered N status, maspin sub-cellular localization and nuclear nm23-H1 expression. The selected variables' accuracy in terms of relapse was 82%. Positive predictive accuracy was 100%, and negative predictive accuracy 79%. Nuclear nm23-H1 expression and maspin pattern, also in association, show promise as recurrence indicators in LSCC. Further studies are needed to shed more light on the nm23-H1 mechanism of action in LSCC and thus find ways to restore nm23-H1 loss. These preliminary findings suggest that re-activating maspin functions might represent an important goal in the treatment of advanced LSCC.

Predictive value of maspin and Ki-67 expression in transurethral resection specimens in patients with T1 bladder cancer.

To evaluate the clinical significance of maspin and Ki-67 expression in patients with newly diagnosed T1 bladder cancer. Maspin and Ki-67 expression was investigated by immunohistochemistry from paraffin-embedded tissues of 68 patients undergoing transurethral resection for bladder cancer. Clinicopathological data were retrospectively reviewed from available charts and pathological reports. Maspin and Ki-67 expression levels were classified according to the staining percentage. Cases in which at least 5% of the tumor cells stained for maspin were scored as positive. Ki-67 labeling index was considered to be positive when samples demonstrated >10% reactivity. Maspin expression was found as an independent predictor of recurrence and progression (P <0.05). Patients with negative maspin expression were 2.191 times more likely to relapse than patients with positive maspin expression. Patients with negative maspin expression were 4.345 times more likely to progress than patients with positive maspin expression. Furthermore, the maspin-negative group was found to have shorter recurrence and progression-free survival (P <0.05). No significant association was found between maspin subcellular localization pattern and recurrence-free, progression-free or overall survival (P >0.05). There was no correlation between Ki-67 expression and tumor recurrence, progression or tumor-related death (P >0.05). Chi-square tests showed a significant relationship between Ki-67 expression and tumor size and tumor grade (P <0.05). Our findings suggested that the evaluation of maspin expression in stage T1 bladder tumors is a useful prognostic marker for predicting the tumor behavior.

Identification of an intrinsic determinant critical for maspin subcellular localization and function.

Maspin, a multifaceted tumor suppressor, belongs to the serine protease inhibitor superfamily, but only inhibits serine protease-like enzymes such as histone deacetylase 1 (HDAC1). Maspin is specifically expressed in epithelial cells and it is differentially regulated during tumor progression. A new emerging consensus suggests that a shift in maspin subcellular localization from the nucleus to the cytoplasm stratifies with poor cancer prognosis. In the current study, we employed a rational mutagenesis approach and showed that maspin reactive center loop (RCL) and its neighboring sequence are critical for maspin stability. Further, when expressed in multiple tumor cell lines, single point mutation of Aspartate346 (D346) to Glutamate (E346), maspinD346E, was predominantly nuclear, whereas wild type maspin (maspinWT) was both cytoplasmic and nuclear. Evidence from cellular fractionation followed by immunological and proteomic protein identification, combined with the evidence from fluorescent imaging of endogenous proteins, fluorescent protein fusion constructs, as well as bimolecular fluorescence complementation (BiFC) showed that the increased nuclear enrichment of maspinD346E was, at least in part, due to its increased affinity to HDAC1. MaspinD346E was also more potent than maspinWT as an HDAC inhibitor. Taken together, our evidence demonstrates that D346 is a critical cis-element in maspin sequence that determines the molecular context and subcellular localization of maspin. A mechanistic model derived from our evidence suggests a new window of opportunity for the development of maspin-based biologically competent HDAC inhibitors for cancer treatment.

Abstract 5203: Identification of an intrinsic determinant critical for maspin subcellular localization and function.

Abstract Abstract: Maspin is a novel serine protease inhibitor (serpin) that does not act as a classical serine protease inhibitor. Our lab demonstrated that maspin directly inhibits the serine protease-like enzyme, histone deacetylase 1 (HDAC1), which is a major deacetylase up-regulated in many types of cancers. To our knowledge, maspin is the only endogenous polypeptide inhibitor of HDAC1 identified thus far. Interestingly, the differential regulation of maspin during tumor progression is characterized by changes in overall expression and subcellular distribution. In particular, a significant shift in maspin subcellular localization from the nucleus to the cytosol has been observed at the transition from benign epithelial cells to precancerous or low grade carcinoma cells. Accumulated clinical evidence suggests that nuclear retention of maspin is correlated with better overall patient survival. We propose that hypothesized that the configuration and conformation of maspin coded by its primary sequence, i.e. cis elements are the key determinants for where maspin may be retained in a given molecular context. We noted that at the C-terminal end of the RCL sequence is an Aspartate 346 (D346) which is also unique among all maspin homologs and orthologs. In the current study, using the mutagenesis approach, we aimed to determine whether D346 acts as the cis element essential for maspin subcellular localization and function. We showed that in comparison to the maspinWT, which is distributed in both cytosol and nuclei of cancer cells, maspinD346E is predominantly localized in the nuclei of cancer cells. In addition, maspinD346E had an increased affinity towards HDAC1 and it was more effective inhibitor of HDAC1 as compared to the maspinWT. These findings led to a novel molecular model that explains how maspin may be disregulated during tumor progression resulting in loss of its tumor suppressive potential. Citation Format: Sijana H. Dzinic, Alexander Kaplun, Xiaohua Li, Margarida M. Bernardo, Ivory Dean, Fulvio Lonardo, Shijie Sheng. Identification of an intrinsic determinant critical for maspin subcellular localization and function. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5203. doi:10.1158/1538-7445.AM2013-5203

Predictive Value of Maspin and Ki-67 Expression in Transurethral Resection Specimens in Patients with T1 Bladder Cancer

To evaluate the clinical significance of maspin and Ki-67 expression in patients with newly diagnosed T1 bladder cancer. Maspin and Ki-67 expression was investigated by immunohistochemistry from paraffin-embedded tissues of 68 patients undergoing transurethral resection for bladder cancer. Clinicopathological data were retrospectively reviewed from available charts and pathological reports. Maspin and Ki-67 expression levels were classified according to the staining percentage. Cases in which at least 5% of the tumor cells stained for maspin were scored as positive. Ki-67 labeling index was considered to be positive when samples demonstrated >10% reactivity. Maspin expression was found as an independent predictor of recurrence and progression (P <0.05). Patients with negative maspin expression were 2.191 times more likely to relapse than patients with positive maspin expression. Patients with negative maspin expression were 4.345 times more likely to progress than patients with positive maspin expression. Furthermore, the maspin-negative group was found to have shorter recurrence and progression-free survival (P <0.05). No significant association was found between maspin subcellular localization pattern and recurrence-free, progression-free or overall survival (P >0.05). There was no correlation between Ki-67 expression and tumor recurrence, progression or tumor-related death (P >0.05). Chi-square tests showed a significant relationship between Ki-67 expression and tumor size and tumor grade (P <0.05). Our findings suggested that the evaluation of maspin expression in stage T1 bladder tumors is a useful prognostic marker for predicting the tumor behavior.

Abstract 2352: Critical structural determinants of maspin necessary for maspin's subcellular localization, biological, and biochemical functions

Abstract Maspin (serpin B5), is the sole member of the serine protease inhibitor superfamily whose homozygous deletion results in embryonic lethality. Furthermore, maspin stands alone as the only non-inhibitory clade B serpin, despite the accumulating evidence for maspin's tumor suppressive effects in the progression of many types of cancer including breast, prostate, and lung. In addition, maspin is exclusively an epithelial-specific serpin with distinctive functions in the nucleus, cytosol, and the extracellular space. Our previous research discovered a novel maspin partnership with serine protease-like molecules such as the zymogen form of urokinase type plasminogen activator (pro-uPA) and histone deacetylase 1 (HDAC1). Structural analyses revealed several distinctive maspin motifs and domains that may be important for partnership of maspin with its target proteins as well as subcellular localization during tumor progression. In this study we aimed to determine if these structural motifs/domains of maspin, in addition to the earlier identified reactive center loop (RCL), are indeed important for maspin complex molecular interactions and biological activities. We utilized the prostate cancer cell line DU145, expressing low endogenous levels of maspin, to deliver wild type maspin and five rational maspin mutants by adenovirus. Different maspin mutants exhibited different affinities for the pro-uPA/uPAR complex on the cell surface and different affinities for intracellular HDAC1 and cytosolic heat shock protein 90 (HSP 90). Based on our biophysical evidence, a unique KDEL motif located downstream of the RCL domain, seems to play a critical role in dictating the subcellular localization and secretion of maspin. It has been shown that the majority of proteins that have a KDEL motif at the extreme C-terminus are endoplasmic reticulum resident proteins. Our study with maspin provides the first evidence that an internal KDEL motif may suffice to direct protein subcellular localization. This finding sheds a new light into the aberrant maspin subcellular localization during tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2352.

MASPIN subcellular localization and expression in oral cavity squamous cell carcinoma

MASPIN, a member of serpin superfamily, has multifaceted biological functions and an unique tumour suppressing activity. Experimental evidences showed that MASPIN suppresses tumour growth, angiogenesis, invasion and metastasis. Only a very limited number of studies considered MASPIN expression in the upper aero-digestive tract carcinomas. It was recently found that nuclear localization of MASPIN was significantly associated with lower recurrence rate and longer disease-free interval in laryngeal carcinoma. The present study investigated the biological and prognostic role of MASPIN in relation to its subcellular localization in oral carcinoma. Sub-cellular pattern of distribution of MASPIN, nuclear and cytoplasmic MASPIN expressions were immunohistochemically determined in 56 consecutive cases of oral carcinoma. Statistical analysis found a significant association between pN-stage and recurrence of disease (P=0.032) and a significantly longer disease-free interval in pN0 patients than in pN+ ones (P=0.038). None of the subcellular expressions of MASPIN was significantly correlated with recurrence of disease and disease-free interval in our series of oral carcinomas. Sixty-one percent of pN0 cases was strongly MASPIN-positive in the cytoplasm of primary carcinoma cells, 33% of the pN+ cases was MASPIN-positive in the cytoplasm. Statistical analysis found a significant association between MASPIN cytoplasmic expression and pN-stage (P=0.032). Negative MASPIN immunoreactivity in carcinoma cells cytoplasm may be useful to identify patients at risk of disease disseminating to neck lymph nodes. Further investigations are necessary to understand the biological role of cytoplasmic MASPIN localization in oral carcinoma.