Loss Of Maspin Expression Research Articles

Abstract 2594: Reactivation of maspin by plant flavone apigenin through inhibition of class I HDACs and increase in p53 transcriptional activity in prostate cancer cells

Abstract Loss of tumor suppressors leads to acquisition of metastatic capability during prostate cancer progression leading to higher mortality. Maspin (SERPINB5) is a unique member of the serpin (serine protease inhibitor) family shown to regulate cell motility, migration and invasiveness. Loss of maspin is frequently identified in clinical prostate cancer specimens and prostate cancer cell lines, therefore novel therapeutic approaches to restore its expression are needed. We recently demonstrated that knockdown of class I HDACs increase maspin expression in prostate cancer cells [Mol Carcinog. 59(8):955-966, 2020]. Apigenin (4', 5, 7-trihydroxyflavone), a plant flavone has shown to possess anticancer properties and alters pathways that regulate tumor cell invasion and metastasis, however, the molecular basis of these effects remains unclear. We investigated whether apigenin has ability to restore maspin expression and contribute to the inhibition of metastasis. Treatment of human prostate cancer LNCaP and 22Rv1 cells, both harboring wild type p53, with 1-40 µM apigenin for 72 h resulted in a dose- and time- dependent decrease in cell invasion and migration with concurrent increase in maspin expression in the nuclear compartment. Since, p53 activates maspin promoter by binding directly to p53 consensus-binding site, we studied the effect of apigenin in potentially restoring p53-mediated maspin levels. Exposure of LNCaP and 22Rv1 cells with 5-20 μM of apigenin resulted in dose-dependent increase in maspin expression and p53 activation through acetylation at the Lys305 residue. These effects were associated with the inhibition of class I HDAC levels. Furthermore, apigenin withdrawal resulted in the loss of maspin expression and p53 acetylation in LNCaP cells. The increased apigenin-mediated p53 acetylation enhanced its binding on maspin promoter, which was associated with decrease in cell invasion and migration. Apigenin treatment also caused accumulation of acetylated histone H3 in total cellular chromatin, increasing accessibility to bind with the promoter sequences of maspin, consistent with the effects elicited by HDAC inhibitor, Tricostatin A. Similar observations of inhibition of class I HDACs and increase in p53 transcriptional activity were noted after feeding apigenin at 20- and 50- µg/day to 22Rv1 tumor xenograft implanted in athymic nude mice. Our results demonstrate that apigenin-mediated increase in maspin expression together with downregulation of class I HDACs, increases p53 activation resulting in decreased invasiveness and migration capabilities in prostate cancer. Citation Format: Eswar Shankar, Albert Lee, Rajnee Kanwal, Sanjay Gupta. Reactivation of maspin by plant flavone apigenin through inhibition of class I HDACs and increase in p53 transcriptional activity in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2594.

Elevated Maspin Expression Is Associated with Better Overall Survival in Esophageal Squamous Cell Carcinoma (ESCC)

Tumor suppressor maspin is a differentially regulated gene in the progression of many types of cancer. While the biological function of maspin in blocking tumor invasion and metastasis is consistent with the loss of maspin expression at the late stage of tumor progression, the differential expression and the biological significance of maspin in early stage of tumor progression appear to be complex and remain to be elucidated. In the current study, we examined the expression of maspin in 84 esophageal squamous cell carcinoma (ESCC) cases (stages I–III) and 55 non-tumor adjacent esophageal tissue specimens by immunohistochemical (IHC) staining. The correlation of maspin with clinicopathological parameters was analyzed. Compared to normal esophageal squamous tissue where 80% (47/55) of the cases expressed maspin at a low to moderate level, all ESCC specimens (100% (84/84)) were positive for maspin expression at a moderate to high level. ESCC with low or moderate maspin expression had significantly shorter postoperative survival rates compared to those that had high maspin expression (p<0.001). Since the correlation of maspin with ESCC histology and the correlation of maspin with ESCC prognosis seem to be at odds, we further investigated the biological function of maspin in ESCC using the established ESCC cell lines. The expression of maspin in five human esophageal squamous cancer cell lines (T12, E450, KYSE150, EC109, and KYSE510) was examined by the Western blot. ESCC cell line KYSE510 that did not express maspin and was stably transfected by maspin cDNA or an empty vector. The resulting transfected cells were characterized in vitro. Maspin expression significantly inhibited cell proliferation, motility and matrigel invasion. Taken together, our data suggest that the transient up-regulation of maspin in the early development of ESCC may be a defense mechanism against further transition towards more malignant phenotypes, ultimately slowing down ESCC tumor progression.

Abstract 486A: Biological characterization of recombinant maspin in breast cancer cells

Abstract Mammary serine protease inhibitor (maspin) is a non-inhibitory member of the serine protease inhibitor (serpin) superfamily and is involved in mammary gland development. Loss of maspin expression is commonly observed during breast cancer progression while re-expression of maspin confers tumor suppressive capabilities. Cellular localization of maspin within the cytosol, nucleus, and association in complexes at the plasma membrane confer different properties in cancer cells adding to the complexity of maspin function. The recombinant form of maspin (rMas) when added to growth media of maspin-null cancer cells recapitulates many of the effects of exogenous maspin re-expression including inhibition of invasion through 3D matrices. These effects highlight the potential of rMas for therapeutic development as a novel adjuvant therapy. The aim of this study was to characterize the uptake, processing and distribution of wild-type and mutated forms of rMas in cancer cells. Using the MDA-MB-231, BT549 and Hs578T breast cancer cell lines we demonstrate that rMas is directly internalized by cancer cells and degraded rapidly. Time-lapse confocal microscopy using Alexa Fluor 594 labeled rMas and nuclear/cytoplasmic extraction of rMas following cellular uptake demonstrate that rMas is predominantly localized and processed in the cytosol. Wild-type rMas inhibited cancer cell invasion through collagen based matrices and this effect was independent of nuclear localization. Interestingly, site-directed mutagenesis of rMas to induce reported polymorphisms (Ile to Val 66, Pro to Ser 176, Leu to Val 187, Ile to Val 319) abrogated the ability of rMas to inhibit invasion. These preliminary experiments contribute to the characterization of rMas following internalization by cancer cells and aid in the therapeutic development of rMas as a potential therapy. Additionally, mutational data from this study support growing evidence that mutations in the maspin gene alter functionality in vitro, and may correlate to clinical outcomes in patients in which maspin is expressed in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 486A. doi:1538-7445.AM2012-486A

Clinical significance of Maspin promoter methylation and loss of its protein expression in invasive ductal breast carcinoma: correlation with VEGF-A and MTA1 expression

Maspin is a serine protease inhibitor with tumor-suppressor activity. Maspin can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. Previous studies indicate that the loss of Maspin expression is closely linked to aberrant methylation of the Maspin promoter. We examined the promoter methylation status of Maspin in tumor and corresponding serum of breast cancer patients. In addition, protein expression of this gene was also assessed to determine possible correlation between promoter hypermethylation and gene silencing. Further, we investigated the correlation of Maspin expression with vascular endothelial growth factor (VEGF-A) and MTA1 expression. Maspin methylation was analyzed by methylation-specific PCR in 100 invasive ductal breast carcinoma patients' tumors and circulating DNA in a prospective study. Promoter hypermethylation was correlated with expression of the encoded protein in tumors by immunohistochemistry. Significant correlation was observed between promoter hypermethylation of Maspin (r = +0.88; p ≤ 0.0001) in tumors and paired sera. Significant association was found between Maspin promoter hypermethylation and loss of its protein expression (p = 0.01, OR = 3.1, 95% CI = 1.3-7.4). The expression of VEGF-A and MTA1 was lower in tumors with high Maspin expression compared to tumors with loss of Maspin expression. Our results indicate that aberrant promoter methylation is associated with loss of Maspin immunoreactivity in breast cancer tissues. Further, loss of Maspin expression is significantly correlated with increased expression of VEGF-A and MTA1.

Maspin expression, angiogenesis, prognostic parameters, and outcome in malignant melanoma

Maspin is a serine protease inhibitor that is thought of as a tumor suppressor because of observations that loss of maspin expression in breast, prostate, and oral cancer is associated with poor prognosis. In addition, maspin may function as an inhibitor of angiogenesis. However, it has been correlated with malignant behavior in pancreatic and ovarian cancer. The role of maspin in malignant melanoma (MM) has not yet been systematically examined. We aimed to examine the immunohistochemical expression of maspin and several proangiogenic factors (vascular endothelial growth factor, transforming growth factor-beta, alphaVbeta3 integrin, cyclooxygenase-2, and CD44) in MM and correlate each to angiogenesis, tumor thickness, and outcome. In all, 77 formalin-fixed, paraffin-embedded MM samples were immunostained for maspin and other proangiogenic factors (vascular endothelial growth factor, transforming growth factor-beta, alphaVbeta3 integrin, cyclooxygenase-2, and CD44) and were correlated with angiogenesis as mean microvessel density. Three normal-appearing skin samples and 10 nevi were also immunostained for maspin. Breslow thickness, Clark level, clinical stage, and follow-up information were obtained for outcome analysis. Immunohistochemical analysis revealed strong nuclear melanocytic maspin expression in all 10 nevi (half of which were dysplastic) but none in melanocytes from 3 normal-appearing skin samples. Strong nuclear maspin staining was demonstrated in 78% of radial phase melanoma and 46% of vertical growth phase melanoma. In addition, there was a significant inverse relationship between maspin and microvessel density (P = .018) and tumor thickness greater than 0.76 mm (P = .007), indicating that maspin is expressed in thinner tumors with less angiogenesis. Conversely, vascular endothelial growth factor expression, Clark level, and Breslow thickness all significantly correlated with microvessel density (P = .047, P = .027, and P = .011, respectively). Cyclooxygenase-2 expression significantly correlated with thicker tumors (P = .006) but not with angiogenesis (P = .714). In addition, Clark level, Breslow thickness, and stage were all significant predictors of overall survival (P < .001, P = .005, and P < .001, respectively). This study represents a single institution. These results demonstrate maspin expression in nevi and radial growth phase melanoma, but this expression seems to be lost in the transition from radial growth phase to vertical growth phase melanoma. In addition, maspin is correlated with decreased angiogenesis and tumor thickness less than 0.76 mm in MM. These results indicate maspin may function as a tumor suppressor in MM.

The maspin expression in canine mammary tumors: an immunohistochemical and molecular study

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.

Maspin and p53 Protein Expression in Gastric Adenocarcinoma and Its Clinical Applications

To investigate the role of maspin and p53 expression in the progression of gastric cancer, and its value as a prognostic indicator. The expression of maspin and p53 in 152 cases of gastric cancer was detected by immunohistochemistry and compared with the clinicopathologic tumor parameters. The relationship between maspin and p53 expression was also analyzed in the gastric cancers. The positive expression rates for maspin and p53 in the cancers were 71.7% (109 of 152 cases) and 56.6% (86 of 152 cases), respectively. Two patterns of immunostaining for maspin were seen in the maspin-positive gastric cancer cases: cytoplasm-only staining (67.0%, 73 of 109 cases) and staining of both cytoplasm and nucleus (33.0%, 36 of 109 cases). Maspin expression showed a negative association with histologic grade, depth of invasion, metastasis, and TNM stage (all P<0.05). p53 expression showed an association with node metastasis, and TNM stage (both P<0.05). Maspin expression was negatively correlated with p53 expression (P<0.001, r=-0.291). In univariate log-rank analysis, loss of maspin expression, histologic grade, distant metastasis, and TNM stage were associated with patient survival. Interestingly, patients with nuclear and cytoplasmic maspin expression survived longer than those with only cytoplasmic expression. However, in multivariate analysis TNM stage and regional node metastasis were the only independent prognostic factors. Maspin expression might be an important factor in tumor progression and patient prognosis, but is not an independent prognostic factor. Maspin expression is inversely correlated with mutant p53 expression in gastric cancer, which suggests that maspin expression is regulated by the p53 pathway.

Loss of Maspin expression is a negative prognostic factor in common salivary gland tumors

Maspin, a 42kDa protein, belongs to the serine protease inhibitor (serpin) family and is suggested to have inhibitory effects on tumor-induced angiogenesis, tumor cell motility, invasion and metastasis and influences prognosis of tumor patients. The aim of the study was to analyze Maspin expression in salivary gland cancer as well as its prognostic impact on survival in comparison to clinical parameters. Immunohistochemical staining was carried out in 73 cases of salivary gland malignancies. High proportions of Maspin expression were observed in adenoid cystic carcinomas, mucoepidermoid carcinomas and carcinomas ex pleomorphic adenoma, low proportions were seen in salivary duct carcinomas. Acinic cell carcinomas did not show any Maspin expression. Analysis of the prognostic impact of Maspin expression was restricted to salivary gland carcinoma types of intermediate malignancy grade (adenoid cystic carcinoma, mucoepidermoid carcinoma and carcinoma ex pleomorphic adenoma). For these tumors, univariate analyses revealed that T-stage (p=0.025), age70 (p=0.0065), loss of Maspin (p=0.0016) and presence of residual tumor (p<0.001) correlated with poor prognosis. In multivariate analysis age70 (p=0.005) and loss of Maspin (p=0.036) were significant prognostic factors. Moreover, negative Maspin staining was associated with lymph node metastasis and residual tumor. According to these findings, Maspin might be useful as a new prognostic marker in adenoid cystic carcinoma and in salivary gland carcinomas with intermediate grade of malignancy where grading systems are still under debate.

Loss of maspin expression correlates with increased invasiveness in malignant melanoma

Loss of maspin expression correlates with increased invasiveness in malignant melanoma

Decreased Immunoreactive Maspin Expression in Intermediate Thickness and Thick Primary Melanoma Lesions

Maspin is a member of the serpin family of protease inhibitors. It is a 42 kDa cytoplasmic protein that is reported to have tumour suppressor activity. The loss of maspin gene expression is correlated with increased invasiveness and the risk of metastases in breast cancer. We studied maspin expression in primary melanoma lesions obtained from 76 patients. Immunostaining of 5 pm sections for maspin expression was obtained using the citrate antigen retrieval method. The extent of immunostaining was scored by recording the proportion of immunoreactive cells and the intensity of immunostaining. Our results demonstrated that maspin expression was down-regulated in intermediate thickness and thick melanoma lesions compared with thin lesions. These results suggest that loss of maspin expression might play a role in melanoma progression, invasion and metastatic dissemination. Further studies are needed to clarify the clinicopathological significance of maspin expression in melanoma.

Elevated nuclear maspin expression is associated with microsatellite instability and high tumour grade in colorectal cancer

Maspin, a member of the serpin family, has been reported to suppress metastasis and angiogenesis in breast and prostate cancers. Overexpression of maspin was associated with adverse prognostic features in several other tumours. In this study, expression of maspin was analysed in 41 colorectal carcinomas with high-frequency microsatellite instability (MSI-H) and 159 microsatellite stable colorectal cancers (MSS/MSI-L) by immunohistochemistry (IHC) and partly by relative quantitative real-time RT-PCR and western blot analyses. Significant upregulation of maspin expression was found in MSI-H tumours compared to both MSS/MSI-L tumours and matched benign colonic mucosa. Increased maspin expression was also found in three MSI-H colon cancer cell lines, but not in three MSS colon cancer cell lines by RT-PCR and western blot analyses. Regulation of maspin expression depended on promoter methylation as tissue specimens and cell lines expressing maspin showed unmethylated maspin promoters, whereas promoter hypermethylation was found in specimens with loss of maspin expression. Intense nuclear maspin immunostaining was seen specifically in MSI-H tumours (p = 0.013), de-differentiated tumours (p = 0.006), and at the invasion front. These findings provide new insights into the role of maspin in colorectal cancer progression and may be useful for diagnosis and treatment strategies.

Loss of maspin expression is associated with development and progression of gastric carcinoma with p53 abnormality

Maspin, a serine protease inhibitor related to the serpin family, was originally identified in normal mammary epithelium. Reduced expression of maspin is related with development, invasion and metastasis of certain human cancers. In the present study, the expression of maspin was examined in gastric mucosa, adenoma and carcinoma by immunohistochemistry and RT-PCR. In non-neoplastic mucosa, maspin was expressed in cytoplasm and cell membrane of foveolar epithelia, fundic glandular cells and pyloric glandular cells. Maspin expression was lost in 71% (71/100) of gastric carcinomas, and in 19% (4/21) of adenomas, respectively. Loss of maspin expression was significantly associated with poorly differentiated histology, advanced stage and deep invasion (P<0.001). There was an inverse correlation between maspin expression and abnormal p53 accumulation. Maspin mRNA expression was lost in all of 8 gastric carcinoma cell lines that was retrieved after treatment with demethylation agent 5-aza-2'-deoxycytidine in 5 of 8 cell lines. These results suggest that loss of maspin expression partly due to DNA methylation may participate in tumor development and progression of gastric carcinoma in relation with p53 pathway. Loss of maspin expression may serve as a biological marker of high-grade malignancy.

Aberrant methylation of the maspin promoter is an early event in human breast cancer.

The maspin gene functions as a tumor suppressor in human breasts, and its expression is frequently lost during breast cancer progression. In vitro models of human breast cancer indicate that the loss of maspin expression is closely linked to aberrant methylation of the maspin promoter. We conducted a study on 30 archival ductal carcinoma in situ (DCIS) specimens to determine if aberrant methylation of the maspin promoter occurred in vivo, and whether it occurred early in breast cancer evolution. Healthy tissue obtained from reduction mammoplasty was used as normal control. Results from immunohistochemical analysis indicate that maspin expression is lost in a substantial fraction of DCIS specimens (57%). Bisulfite sequencing of DNA isolated from laser capture-microdissected normal and neoplastic ducts showed that loss of maspin expression was often, but not always, linked to aberrant methylation of the maspin promoter, suggesting that other mechanisms, in addition to aberrant methylation, participate and/or cooperate to silence maspin gene expression. Taken together, these results indicate that aberrant methylation of the maspin promoter is an early event in human breast cancer.

Loss of maspin expression in prostate cancer induced by a P53- and HER-2- pathway/overexpression profiling by whole-mount derived tissue microarrays

Loss of maspin expression in prostate cancer induced by a P53- and HER-2- pathway/overexpression profiling by whole-mount derived tissue microarrays

Association of maspin expression with the high histological grade and lymphocyte-rich stroma in early-stage breast cancer.

Maspin is a recently described member of the serpin family or protease inhibitors that is known to be a tumour suppressor gene product. Loss of maspin expression has been found in most breast cancer cases and is correlated with cell motility and tumour invasiveness. However, its precise role in human breast cancer remains to be discovered. We aimed to evaluate the role of maspin in early-stage breast cancer. We analysed the expression of maspin in 192 stage I and II primary breast cancers by immunohistochemistry. Of these cases, 34.4% showed maspin expression. Maspin expression was more frequently found in invasive ductal carcinoma (36.4%) than in invasive lobular carcinoma (7.1%). High maspin expression was demonstrated in breast cancers showing high histological grade or lymphocyte-rich stroma (P < 0.05). Maspin expression was not associated with overall and disease-free survival rate of breast cancer. The results indicate that different biological mechanisms may be responsible for maspin expression in histologically distinct types of breast cancer. Our survey suggests that maspin expression in breast cancer might have a compensatory role rather than prognostic one.

Maspin expression inversely correlates with breast tumor progression in MMTV/TGF-alpha transgenic mouse model.

Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive activity. To date, despite the mounting evidence implicating the potential diagnostic/prognostic and therapeutic value of maspin in breast and prostate carcinoma, the lack of a suitable animal model hampers the in vivo investigation on the role of maspin at different stages of tumor progression. In this study, we used MMTV/TGF-alpha transgenic mouse model to study the expression profile of maspin in mammary tumor progression. Histopathological examinations of MMTV/TGF-alpha transgenic mice revealed TGF-alpha expression leading to hyperproliferation, hyperplasia, and occasional carcinoma in mammary gland. Interestingly, when MMTV/TGF-alpha transgenic mice were breed to homozygocity, they also developed characteristic skin papillomas. Immunohistochemistry analysis of maspin expression in the breast tissues of TGF-alpha transgenic mice showed a direct correlation between down-regulation of maspin expression and tumor progression. The loss of maspin expression was concomitant with the critical transition from carcinoma in situ to invasive carcinoma. Subsequent in-situ hybridization analyses suggest that the down-regulation of maspin expression is primarily a transcriptional event. This data is consistent with the tumor suppressive role of maspin. Furthermore, our data suggests that MMTV/TGF-alpha transgenic mouse model is advantageous for in vivo evaluation of both the expression and the biological function of maspin during the slow multi-stage carcinogenesis of mammary gland.

Expression of maspin in prostate cells is regulated by a positive ets element and a negative hormonal responsive element site recognized by androgen receptor.

Prostate cancer is the most common cancer in men. The molecular mechanisms leading to its development are poorly understood. Maspin is a tumor-suppressing serpin expressed in normal breast and prostate epithelium. We have found that expression of maspin in normal and carcinoma-derived prostate epithelial cells is differentially regulated at the transcriptional level. We have identified two different kinds of cis elements, Ets and hormonal responsive element (HRE), in the maspin promoter. The Ets element is active in regulating maspin expression in normal prostate epithelial cells but inactive in tumor cells. The HRE site is a negative element that is active in both cell types. This negative DNA sequence can repress a heterologous promoter recognized by the androgen receptor. We conclude that expression of maspin is under the influence of both a positive Ets and a negative HRE element. Loss of maspin expression during tumor progression apparently results from both the absence of transactivation through the Ets element and the presence of transcription repression through the negative HRE element recognized by androgen receptor.