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Identification of different maspin forms in MCF-10A cells. Whole cell extracts were analyzed by 2D-SDS-PAGE followed by Western blot with anti-maspin. Isolectric focalizations were carried out in three different IPG strips: (A) 100 lg protein/11 cm pH 3-10; (B) 700 lg/13 cm pH 4-7 and (C) 2000 lg/17 cm pH 4.7-5.9. Arrows and arrowhead indicate different maspin forms.
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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...
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... residues under our experimental condition. Since addition of a phosphate group adds a negative charge to the protein, we reasoned we would be able to detect different mas- pin forms by IEF followed by Western blot. For this purpose 100 lg of whole cell extract were analyzed on an 11 cm pH 3-10 linear gradient IPG strip. A predominant spot ( Fig. 2A, arrow) next to a possible precursor of an independent spot ( Fig. 2A, arrowhead) was detected with anti-maspin. By this first analysis maspin pI was estimated to be around 5.65, which approaches a previous prediction [8]. In a second analysis 700 lg was applied on a 13 cm IPG strip with a narrower pH gradient (pH 4-7). Two distinct spots ...
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... group adds a negative charge to the protein, we reasoned we would be able to detect different mas- pin forms by IEF followed by Western blot. For this purpose 100 lg of whole cell extract were analyzed on an 11 cm pH 3-10 linear gradient IPG strip. A predominant spot ( Fig. 2A, arrow) next to a possible precursor of an independent spot ( Fig. 2A, arrowhead) was detected with anti-maspin. By this first analysis maspin pI was estimated to be around 5.65, which approaches a previous prediction [8]. In a second analysis 700 lg was applied on a 13 cm IPG strip with a narrower pH gradient (pH 4-7). Two distinct spots could be detected, a strong one and a more acidic less abun- dant spot (Fig. ...
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... 2A, arrowhead) was detected with anti-maspin. By this first analysis maspin pI was estimated to be around 5.65, which approaches a previous prediction [8]. In a second analysis 700 lg was applied on a 13 cm IPG strip with a narrower pH gradient (pH 4-7). Two distinct spots could be detected, a strong one and a more acidic less abun- dant spot (Fig. 2B, arrow and arrowhead, respectively). In order to increase the relative focusing power and reach the finest possible resolution, 2 mg of whole cell extract were applied to a 17 cm strip with a micro-range gradient (pH 4.7-5.9). Four different spots were detected (Fig. 2C, arrows). In order to confirm these spots were due to phosphorylation, maspin was immunoprecipitated ...
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... spots could be detected, a strong one and a more acidic less abun- dant spot (Fig. 2B, arrow and arrowhead, respectively). In order to increase the relative focusing power and reach the finest possible resolution, 2 mg of whole cell extract were applied to a 17 cm strip with a micro-range gradient (pH 4.7-5.9). Four different spots were detected (Fig. 2C, arrows). In order to confirm these spots were due to phosphorylation, maspin was immunoprecipitated from whole cell extract and immunoprecipitated material was ana- lyzed by 2D-SDS-PAGE followed by Western blot with anti-phos- photyrosine. In at least four different attempts using different antibodies, we were never able to detect maspin spots ...
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... which is compatible with both IEF and acidic phosphatase treatment (de- scribed in Section 2). Two milligrams of nuclear extract was ana- lyzed by 2D-SDS-PAGE followed by Western blot with anti- maspin. Four maspin spots were detected in this fraction (Fig. 3A), which present a similar distribution pattern observed in whole cell extract (Fig. 2C). Acidic phosphatase treatment of the extract followed by similar analysis completely abolished three of the four spots initially detected (Fig. 3B), indicating that these three spots are phosphorylated maspin. To further support the phosphorylation of maspin forms and confirm that it occurs on tyrosine residues, MCF-10A cells were ...
Citations
... In line with these observations, we detected a reduced expression of SER-PINB5 in DPM1 KD HaCaT keratinocytes by western blot and immunofluorescence-based assays, while SERPINB5 transcript levels were unchanged (Fig. S2, h-l), suggesting posttranslational regulation ( Fig. S2 l). EGFR (epidermal growth factor receptor)dependent signaling mediates SERPINB5 phosphorylation and modulates its localization in the cell (Tamazato Longhi and Cella, 2012;Tamazato Longhi et al., 2016). Interestingly, the membrane localization of EGFR in sgDPM1 cells was perturbed and EGFR showed reduced activity, as indicated by phosphorylation at Y1068 (Fig. 3, g-i). ...
Glycosylation is essential to facilitate cell–cell adhesion and differentiation. We determined the role of the dolichol phosphate mannosyltransferase (DPM) complex, a central regulator for glycosylation, for desmosomal adhesive function and epidermal differentiation. Deletion of the key molecule of the DPM complex, DPM1, in human keratinocytes resulted in weakened cell–cell adhesion, impaired localization of the desmosomal components desmoplakin and desmoglein-2, and led to cytoskeletal organization defects in human keratinocytes. In a 3D organotypic human epidermis model, loss of DPM1 caused impaired differentiation with abnormally increased cornification, reduced thickness of non-corneal layers, and formation of intercellular gaps in the epidermis. Using proteomic approaches, SERPINB5 was identified as a DPM1-dependent interaction partner of desmoplakin. Mechanistically, SERPINB5 reduced desmoplakin phosphorylation at serine 176, which was required for strong intercellular adhesion. These results uncover a novel role of the DPM complex in connecting desmosomal adhesion with epidermal differentiation.
... Maspin can be found in epithelial structures and also in non-epithelial ones [3]. In pathology practice, it is used to mark squamous epithelium, urothelium, basal cells, fibroblasts, myoepithelial cells, mucosa of the gastrointestinal tract, and endothelial cells, mainly in the cytoplasm, in secretory vesicles and in the cell membrane [1,2,4,[6][7][8][9]. In tumor pathology, maspin is a tumor suppressor that inhibits invasion and angiogenesis and also regulates apoptosis [3,7,8], but it can also present oncogenic activity depending on tumor location and histology [6] and on the subcellular maspin localization [3,8,9]. ...
... In pathology practice, it is used to mark squamous epithelium, urothelium, basal cells, fibroblasts, myoepithelial cells, mucosa of the gastrointestinal tract, and endothelial cells, mainly in the cytoplasm, in secretory vesicles and in the cell membrane [1,2,4,[6][7][8][9]. In tumor pathology, maspin is a tumor suppressor that inhibits invasion and angiogenesis and also regulates apoptosis [3,7,8], but it can also present oncogenic activity depending on tumor location and histology [6] and on the subcellular maspin localization [3,8,9]. Both in gastric carcinomas and colorectal carcinomas (CRC), cytoplasmic immunohistochemical maspin positivity is considered to be an indicator for low metastatic risk and for late recurrence, but nuclear immunohistochemical positivity is related to early recurrence, especially in advanced stage carcinomas [8,[10][11][12][13][14][15][16][17][18][19]. ...
Background: Gastrointestinal adenocarcinomas are a worldwide and some of the most
important causes of death related to cancers. MLH1, PMS2, and K-Ras are some of the main
molecules responsible for the control of cellular proliferation. They are widely used as biomarkers
for the evaluation of the features of tumoral processes and the clinicopathological characteristics.
They depend on the type of cells implied in the tumoral process, and it can be observed in the
concentrations of them in different biological fluids. Maspin, also known as peptidase inhibitor
5 or serpin B5 is a tumor suppressor which inhibits invasion and angiogenesis and also regulates
apoptosis, but it can also present oncogenic activity depending on tumor location and histology
and on the subcellular maspin localization. Its correlations with gastric and colorectal carcinomas
have been emphasized in a series of articles, and in this work, a method is used to quantify the
concentrations of maspin in three biological fluids, allowing correlations with pathological features.
Methods: Patients with their clinical and pathological features were selected from the database of the
project GRAPHSENSGASTROINTES and used accordingly with the Ethics committee approval nr.
32647/2018 awarded by the County Emergency Hospital from Targu-Mures. Three kinds of samples
have been analyzed (saliva, whole blood, and urine) using a stochastic method using stochastic
microsensors. Results: The results obtained using stochastic sensors were correlated with the location
of cancer, and there have been elaborated a series of criteria to differentiate gastric cancers from
colorectal ones. Conclusions: There can be differentiation between the two types of cancers by using
the concentrations of MLH1, PMS2, and K-Ras in saliva and urine samples or the levels of maspin
in whole blood and urine or in whole blood, urine, and saliva. The data analysis led to a series of
criteria for evaluation of the cancer location. Using only MLH1 and PMS2 concentrations in one of
the two kinds of samples was only indicative and did not cover most cases. The use of the criteria
only for MLH1 and PMS2 increased the probability of finding out the location, but the best results
require the concentrations of K-Ras in the two kinds of samples as additional criteria.
... A review published in 2019 by Banias et al. showed that, in normal organs and tissues, maspin can be present in epithelial and non-epithelial structures [6]. It can mark urothelium, squamous epithelium, basal cells of the prostate and bronchial epithelium, placental cyto-and syncytio-trophoblasts, fibroblasts, myoepithelial cells of the mammary gland, endometrium, mucosa of the gastrointestinal tract (esophagus, stomach, small intestine, colon and rectum), testis, thymus, as well as corneal keratocytes, epithelial and endothelial cells [4,5,7,[9][10][11][12]. Any human tissue might express maspin, mainly in the cytoplasm, secretory vesicles and cell membrane but the expression level is not similar [4,9]. ...
... It can also have oncogenic activity and its role depends on the tumor histology and tumor localization [9]. Besides the tissue-specificity, the role of maspin gene also depends on the subcellular localization of the maspin protein [2,6,11,12]. Because sodium peroxidovanadate, a tyrosine phosphatase inhibitor, induced experimentally upregulation of cytoplasmic maspin, it is presumed that subcellular localization is probably regulated by maspin tyrosine phosphorylation [11]. ...
... In early stages, nuclear maspin might be related with risk for lymph node metastases [27]. Decreased maspin increases the risk of tumor progression and occurrence of distant metastases [11,12,16,24,25]. In CRC maspin is even described as an immunogen or "autoantibody-inducing autoantigen" with immunomodulatory role [4,28,29]. ...
Simple Summary
In this article we have highlighted the possible role of nuclear maspin in identification of tumor cells “on the point of budding” and the epithelial mesenchymal transition phenomenon of these cells, along with a deep exploration of the maspin-molecular mediated mechanisms in colorectal cancer.
Abstract
In this review the authors aimed to emphasize the practical value of nuclear expression of the mammary serine protease inhibitor (maspin), also known as serpin B5 protein, in colorectal carcinoma (CRC), from pre-malignant disorders to carcinogenesis and metastasis. As the role of maspin is controversial and not yet understood, the present update highlights the latest data revealed by literature which were filtrated through the daily experience of the authors, which was gained at microscopic examination of maspin expression in CRCs and other tumors for daily diagnosis. Data regarding the subcellular localization of maspin, in correlation with the microsatellite status, grade of tumor dedifferentiation, and epithelial-mesenchymal transition (EMT) phenomenon of the tumor buds were presented with details. An original observation refers to the maspin capacity to mark the tumor cells which are “at the point of budding” that were previously considered as having “hybrid EMT phenotype”. It refers to the transitional status of tumor cell that is between “epithelial status” and “mesenchymal status”. The second original hypothesis highlights the possible role of maspin in dysregulating the intestinal microbiota, in patients with idiopathic inflammatory bowel diseases (IBD) and inducing IBD-related CRC. The dynamic process of budding and EMT of tumor buds, possible mediated by maspin, needs further investigation and validation in many human CRC samples. The histological and molecular data reveal that synthesis of maspin-based therapeutics might represent a novel individualized therapeutic strategy for patients with CRC.
... The observation that maspin can diffuse into the nucleus does not exclude the possibility that a regulated mechanism takes place. We have previously reported the existence of different maspin isoforms in the cell [22,32]. Maspin is found in different cellular compartments other than the nucleus, including mitochondria [33], endoplasmic reticulum-associated vesicles [2], plasma membrane [34] and exosomes [35]. ...
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 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 the maspin putative NLS is partially hidden in maspin 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.
... The observation that maspin can diffuse to the nucleus does not exclude the possibility that a regulated mechanism takes place. We have previously reported the existence of different maspin isoforms in the cell [16,22]. Maspin is found in different cellular compartments other than nucleus, including mitochondria [23], endoplasmic reticulum-associated vesicles [2], plasma membrane [24] and exosomes [25]. ...
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 suggest that subcellular localization plays an essential role on maspin tumor suppression activity. In this study we investigated the molecular mechanisms underlying maspin nucleocytoplasmic shuttling. An in vitro nuclear-import assay using digitonin-permeabilized HeLa cells demonstrated that maspin enters the nucleus by an energy- and carrier-independent mechanism. However, previous studies indicated that maspin subcellular localization is regulated in the cell. Using a nuclear localization signal (NLS) prediction software, we identified a putative NLS in the maspin amino acid sequence. To distinguish between passive and regulated nuclear translocation, maspinNLS or the full-length protein (MaspinFL) were fused to 5GFP, rendering the construct too large to enter the nucleus passively. Unexpectedly, 5GFP-maspinNLS, but not maspinFL-5GFP, entered the nucleus of HeLa cells. Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N, suppressed 5GFP-maspinNLS nuclear localization. In summary, we provide evidence that maspin translocates to the nucleus passively. In addition, we identified a peptide in the maspin protein sequence, which is able to drive a 5GFP construct to the nucleus in an energy-dependent manner.
... Maspin inhibits cell motility by suppressing Rac1 and PAK1 activity and promotes cell adhesion via the PI3K/ERK pathway [28]. In addition, EGFR signaling promotes maspin phosphorylation and nuclear localization, where it inhibits gene transcription [29]. Although our results showed that resistance to AZD9291 partially arise through maspin, further study is needed to determine how maspin is involved in AZD9291 resistance. ...
AZD9291 (osimertinib) is approved for standard care in patients with EGFR T790M-positive non-small cell lung cancer (NSCLC) after prior EGFR TKI progression. Furthermore, AZD9291 is now being evaluated as a first-line treatment for NSCLC patients with activation EGFR mutations. Based on previous experiments, resistance to AZD9291 as a first-line treatment may also emerge. Thus, identification and understanding of resistance mechanisms to AZD9291 as a first-line treatment can help direct development of future therapies. AZD9291-resistant cells (PC9/AZDR) were established using EGFR inhibitor-naïve PC9 cells. Resistance mechanisms were analyzed using next-generation sequencing (NGS) and a proteome profiler array. Resistance to AZD9291 developed through aberrant activation of ERK signaling by an EGFR-independent mechanism. The combination of a MEK inhibitor with AZD9291 restored the sensitivity of PC9/AZDR cells in vitro and in vivo. PC9/AZDR cells also showed increased MET expression and an HRAS G13R mutation. In addition, maspin expression was higher after AZD9291 treatment in PC9/AZDR cells. Sustained ERK activation confers resistance to AZD9291 as a first-line therapy. Thus, co-targeting EGFR and MEK may be an effective strategy to overcome resistance to AZD9291.
... These data underscore the importance of understanding how SerpinB5 is regulated in a non-transformed model as well as in a physiological context. SerpinB5 phosphorylation has been identified in mammary and corneal epithelial cell lines [20][21][22]. Whether this modification occurs in vivo and the signaling pathways involved remain to be elucidated. In this study we characterized SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and investigated the underlying signaling pathway in the non-transformed MCF-10A model system, which expresses SerpinB5 endogenously. ...
... SerpinB5 post-translational modifications have been previously reported in different immortalized and transformed cell lines, including cysteine S-nitrosylation [30], phosphorylation on serine, threonine and tyrosine residues [20][21][22] and acetylation [31,32]. However, the biological significance and the presence of these modifications in vivo and in different developmental stages have not been addressed. ...
... However, the biological significance and the presence of these modifications in vivo and in different developmental stages have not been addressed. Since we have previously identified three different Ser-pinB5 phosphoforms in MCF-10A cells, a non-transformed human mammary epithelial cell line [21], we were interested in looking at SerpinB5 phosphorylation in the mammary gland. For this purpose, mammary glands from female mice at days 1, 10 and 20 of lactation and days 1 and 5 of involution were isolated, protein extracts were prepared and analyzed by Phos-Tag™ SDS-PAGE followed by immunoblot with anti-SerpinB5. ...
Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally regulated. In vitro analyses indicate that SerpinB5 phosphorylation is regulated by EGFR ligands, but EGF appears to be the only able to induce SerpinB5 nuclear localization.
... Furthermore, four serpins (C0LF32, H9AXB3, Q41593, and Q9ST57) were found to be phosphorylated (Additional file 5: Table S3-C) and all had one phosphorylated site at Thr7 in the N-terminus ( Figure 6). Recent studies have demonstrated that the phosphorylation of tyrosine in maspins, which belong to the serpin family, plays a role in increasing maspin expression and accumulation in the cytoplasm [68]. The phosphorylation at Thr24 at the C-terminal 26-residue peptide can significantly increase the activity of serpin A1 [69]. ...
... Khalkhali-Ellis et al. [8] reported that secretory maspin could deposit in the extracellular milieu and be incorporated into the matrix for tissue remodeling to suppress invasion. Tamazato et al. [9] demonstrated that EGFR signaling promoted maspin phosphorylation and nuclear localization, where it inhibited gene transcription directly or via histone deacetylase 1, including CSF-1, Bax, cytokeratin 18, and p21 [10][11][12]. ...
Maspin is a mammary serine protease inhibitor that is encoded by human SERPINB5 gene, and inhibits invasion and metastasis of cancer cells as a tumor suppressor. We performed a systematic meta- and bioinformatics analysis through multiple online databases up to Feb 10, 2017. We found down-regulated maspin expression in gastric cancer, compared with normal mucosa and dysplasia (p < 0.05). Maspin expression was negatively correlated with depth of invasion, TNM staging and dedifferentiation of gastric cancer (p < 0.05). Nuclear maspin expression was higher in intestinal- than diffuse-type carcinoma (p < 0.05). An inverse association between maspin expression and unfavorable overall survival was found in patients with gastric cancer (p < 0.005). According to bioinformatics databases, SERPINB5 mRNA expression was higher in gastric cancer than normal tissues (p < 0.05), and negatively correlated with depth of invasion, TNM staging and dedifferentiation of gastric cancer (p < 0.05). According to KM plotter, we found that a higher SERPINB5 expression was positively correlated with overall and progression-free survival rates of all cancer patients, even stratified by aggressive parameters (p < 0.05). These findings indicated that maspin expression might be employed as a potential marker to indicate gastric carcinogenesis, subsequent progression, and even prognosis.
... Furthermore, four serpins (C0LF32, H9AXB3, Q41593, and Q9ST57) were found to be phosphorylated (Additional file 5: Table S3-C) and all had one phosphorylated site at Thr7 in the N-terminus ( Figure 6). Recent studies have demonstrated that the phosphorylation of tyrosine in maspins, which belong to the serpin family, plays a role in increasing maspin expression and accumulation in the cytoplasm [68]. The phosphorylation at Thr24 at the C-terminal 26-residue peptide can significantly increase the activity of serpin A1 [69]. ...
Wheat (Triticum aestivum L.) is an economically important grain crop. Two-dimensional gel-based approaches are limited by the low identification rate of proteins and lack of accurate protein quantitation. The recently developed isobaric tag for relative and absolute quantitation (iTRAQ) method allows sensitive and accurate protein quantification. Here, we performed the first iTRAQ-based quantitative proteome and phosphorylated proteins analyses during wheat grain development.
The proteome profiles and phosphoprotein characterization of the metabolic proteins during grain development of the elite Chinese bread wheat cultivar Yanyou 361 were studied using the iTRAQ-based quantitative proteome approach, TiO2 microcolumns, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among 1,146 non-redundant proteins identified, 421 showed at least 2-fold differences in abundance, and they were identified as differentially expressed proteins (DEPs), including 256 upregulated and 165 downregulated proteins. Of the 421 DEPs, six protein expression patterns were identified, most of which were up, down, and up-down expression patterns. The 421 DEPs were classified into nine functional categories mainly involved in different metabolic processes and located in the membrane and cytoplasm. Hierarchical clustering analysis indicated that the DEPs involved in starch biosynthesis, storage proteins, and defense/stress-related proteins significantly accumulated at the late grain development stages, while those related to protein synthesis/assembly/degradation and photosynthesis showed an opposite expression model during grain development. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 12 representative genes encoding different metabolic proteins showed certain transcriptional and translational expression differences during grain development. Phosphorylated proteins analyses demonstrated that 23 DEPs such as AGPase, sucrose synthase, Hsp90, and serpins were phosphorylated in the developing grains and were mainly involved in starch biosynthesis and stress/defense.
Our results revealed a complex quantitative proteome and phosphorylation profile during wheat grain development. Numerous DEPs are involved in grain starch and protein syntheses as well as adverse defense, which set an important basis for wheat yield and quality. Particularly, some key DEPs involved in starch biosynthesis and stress/defense were phosphorylated, suggesting their roles in wheat grain development.
