Yanhua Xuan

Expression of Indian Hedgehog signaling molecules in breast cancer.

PurposeTo investigate the clinicopathological significance and expression pattern of Hedgehog (Hh) signaling molecules in breast normal glands and invasive ductal carcinoma.Materials and methodsA total of 142 cases, including 21 of normal breast and 121 of invasive ductal carcinoma of the breast, were immunohistochemically analyzed for Ihh, Ptch, Smo, Gli-1, Gli-2, and Gli-3 protein expression.ResultsAll of Hh signaling molecules were greatly enhanced in invasive ductal carcinoma compared with the normal breast epithelia. The expressions of Ihh, Smo, and Gli-2 were increased in PR negative cases, and the expressions of Ihh, Ptch, and Gli-1/2/3 were statistically correlated with increased proliferating index of Ki-67 in invasive ductal carcinoma. Ihh and Gli-1/2/3 expressions were correlated with node metastasis. Additionally, the protein expressions of Ihh, Ptch, and Gli-2 were correlated with the clinical stage of breast cancer.ConclusionsHedgehog signaling molecules play an important role in the progression of invasive ductal carcinoma of breast.

Tenascin-C, a Prognostic Determinant of Esophageal Squamous Cell Carcinoma

Background Tenascin-C, an adhesion modulatory extracellular matrix molecule, is highly expressed in numerous human malignancies; thus, it may contribute to carcinogenesis and tumor progression. We explored the clinicopathological significance of Tenascin-C as a prognostic determinant of esophageal squamous cell carcinoma (ESCC). Methods In ESCC patient tissues and cell lines, the presence of isoforms were examined using western blotting. We then investigated Tenascin-C immunohistochemical expression in 136 ESCC tissue samples. The clinical relevance of Tenascin-C expression and the correlation between Tenascin-C expression and expression of other factors related to cancer-associated fibroblasts (CAFs) were also determined. Results Both 250 and 350 kDa sized isoforms of Tenascin-C were expressed only in esophageal cancer tissue not in normal tissue. Furthermore, both isoforms were also identified in all of four CAFs derived from esophageal cancer tissues. Tenascin-C expression was remarkably higher in ESCC than in adjacent non-tumor esophageal epithelium (p < 0.001). Tenascin-C expression in ESCC stromal fibroblasts was associated with patient’s age, tumor (pT) stage, lymph node metastasis, clinical stage, and cancer recurrence. Tenascin-C expression in cancer cells was correlated with an increase in tumor-associated macrophage (TAM) population, cancer recurrence, and hypoxia inducible factor1α (HIF1α) expression. Moreover, Tenascin-C overexpression in cancer cells and stromal fibroblasts was an independent poor prognostic factor for overall survival (OS) and disease-free survival (DFS). In the Cox proportional hazard regression model, Tenascin-C overexpression in cancer cells and stromal fibroblasts was a significant independent hazard factor for OS and DFS in ESCC patients in both univariate and multivariate analyses. Furthermore, Tenascin-C expression in stromal fibroblasts of the ESCC patients was positively correlated with platelet-derived growth factor α (PDGFRα), PDGFRβ, and smooth muscle actin (SMA) expression. The 5-year OS and DFS rates were remarkably lower in patients with positive expressions of both Tenascin-C and PDGFRα (p < 0.001), Tenascin-C and PDGFRβ (p < 0.001), Tenascin-C and SMA (p < 0.001), Tenascin-C and fibroblast activation protein (FAP) (p < 0.001), and Tenascin-C and fibroblast-stimulating protein-1 (FSP1) (p < 0.001) in ESCC stromal fibroblasts than in patients with negative expressions of both Tenascin-C and one of the abovementioned CAF markers. Conclusion Our results show that Tenascin-C is a reliable and significant prognostic factor in ESCC. Tenascin-C may thus be a potent ESCC therapeutic target.

Twist1 is highly expressed in cancer-associated fibroblasts of esophageal squamous cell carcinoma with a prognostic significance

Cancer-associated fibroblasts (CAFs) play important roles in cancer progression. Twist1 was recently reported to be a key regulator of CAFs in gastric cancer, but its role in other types of cancer remains unclear, especially for esophageal squamous cell carcinoma (ESCC). We assessed the Twist1 expression on stromal fibroblasts using immunohistochemistry in 169 tissue specimens from ESCC patients, and performed in vitro and in vivo experiments to confirm the role of Twist1 in CAFs of ESCC. And we investigated the biological pathways that are activated in Twist1-high ESCC using The Cancer Genome Atlas (TCGA) data. The expression of Twist1 in stromal fibroblasts was observed in 89.9% of ESCC patients and positively associated with the increased depth of tumor invasion, lymph node metastasis, and advanced clinical stage, and a significant adverse prognostic factor in overall survival. Twist1-expressing stromal fibroblasts also expressed representative CAF markers, and co-localization of Twist1 and CAF markers were confirmed by confocal immunofluorescence imaging. Bioinformatic analysis of mRNA expression data of esophageal cancer from TCGA revealed that gene sets of CAFs were highly enriched in Twist1-high ESCC. Depletion of Twist1 in ex vivo cultured ESCC CAFs induced significant decrease in migration, invasion, colony formation, sphere formation, and contractibility of ESCC cancer cells compared to control CAFs. Furthermore, Twist1-expressing fibroblasts remarkably enhanced the in vivo tumorigenicity of ESCC in a xenograft model. In conclusion, Twist1 could be a novel CAF marker for the prognostic evaluation of ESCC patients as well as a potent therapeutic target for ESCC.Cancer-associated fibroblasts (CAFs) play important roles in cancer progression. Twist1 was recently reported to be a key regulator of CAFs in gastric cancer, but its role in other types of cancer remains unclear, especially for esophageal squamous cell carcinoma (ESCC). We assessed the Twist1 expression on stromal fibroblasts using immunohistochemistry in 169 tissue specimens from ESCC patients, and performed in vitro and in vivo experiments to confirm the role of Twist1 in CAFs of ESCC. And we investigated the biological pathways that are activated in Twist1-high ESCC using The Cancer Genome Atlas (TCGA) data. The expression of Twist1 in stromal fibroblasts was observed in 89.9% of ESCC patients and positively associated with the increased depth of tumor invasion, lymph node metastasis, and advanced clinical stage, and a significant adverse prognostic factor in overall survival. Twist1-expressing stromal fibroblasts also expressed representative CAF markers, and co-localization of Twist1 and CAF markers were confirmed by confocal immunofluorescence imaging. Bioinformatic analysis of mRNA expression data of esophageal cancer from TCGA revealed that gene sets of CAFs were highly enriched in Twist1-high ESCC. Depletion of Twist1 in ex vivo cultured ESCC CAFs induced significant decrease in migration, invasion, colony formation, sphere formation, and contractibility of ESCC cancer cells compared to control CAFs. Furthermore, Twist1-expressing fibroblasts remarkably enhanced the in vivo tumorigenicity of ESCC in a xenograft model. In conclusion, Twist1 could be a novel CAF marker for the prognostic evaluation of ESCC patients as well as a potent therapeutic target for ESCC.

Tenascin C is a prognostic determinant and potential cancer-associated fibroblasts marker for breast ductal carcinoma

Tenascin C (TNC) is a key of extracellular matrix glycoprotein and highly express in numerous human malignancies. Herein, we attempted to clarify the clinicopathological significance of TNC as a prognostic determinant of breast ductal carcinoma. Then, we investigated TNC immunohistochemical expression in 150 breast ductal carcinomas and 27 normal breast tissue samples. Clinical relevance of TNC expression and the association TNC expression with other factors related to cancer-associated fibroblasts were also examined. In results, TNC expression was significantly higher in breast ductal carcinoma (56.0%) than normal breast tissues (25.9%). The upregulation TNC in cancer stromal were associated with pT stage (P=0.003), lymph node metastasis (P=0.002) and tumor node metastasis stage (P=0.001), also was correlated with an increase in tumor-associated macrophage population (P<0.001). The microvessel density (MVD) was significantly higher in TNC positive group than in negative group (P<0.001). In both univariate and multivariate Cox regression analyses, TNC was an independent poor prognostic factor for overall survival (OS) in breast ductal carcinoma patients. Importantly, over-expression TNC (P<0.001), FSP1 (P<0.001), SMA (P=0.002) and Vimentin (P=0.049) were significantly correlation with the lower OS (P<0.005). In addition, TNC expression in breast ductal carcinoma stromal was positively correlated with FSP1 (P<0.001), SMA (P=0.001) and Vimentin (P<0.001). In conclusion, the high expression of TNC could be a useful cancer-associated fibroblasts marker for the prediction of prognosis of breast ductal carcinoma patients.

Gli1 expression in cancer stem-like cells predicts poor prognosis in patients with lung squamous cell carcinoma

PURPOSE Glioma-associated oncogene homolog 1 (Gli1) is involved in cancer stem cell (CSC) maintenance in various tumors; however, its expression and clinical significance in lung squamous cell carcinoma (LSCC) has not been reported. In this study, we aimed to reveal the clinical significance of Gli1 in LSCC and investigate the potential of Gli1 as a CSC marker by comparing its expression with that of other stemness-related genes in LSCC. METHODS We assessed the expressions of Gli1, LSD1, CD44, Sox9 and Sox2 by immunohistochemistry in the tissue specimens obtained from 101 patients with LSCC. The relationship of Gli1 expression with clinicopathological parameters and cell-cycle regulating genes was investigated. RESULTS Gli1 expression was significantly correlated with T stage (P<0.001), lymph node metastasis (P=0.002), and clinical stage (P=0.005) of LSCC. The Kaplan-Meier survival analysis revealed that the expression of Gli1 in LSCC was all significantly associated with poor overall survival (OS: P=0.005). Cox regression analysis further confirmed that Gli1 is a prognostic marker of unfavorable clinical outcome of LSCC. Gli1 expression was significantly correlated with the expression of stemness-related genes such as LSD1 (P=0.009) and CD44 (P<0.001), but not with those of Sox2 and Sox9. However, Gli1 expression was associated with the expression of hypoxia-inducible factors1α (HIF1α; P<0.001) and Cyclin D1 (P=0.002), respectively. In additionally, microvessel density (MVD) was significantly higher in Gli1-positive LSCC than in the negative LSCC (P=0.026). CONCLUSIONS Our results suggest that Gli1 may be a potential LSCC stem cell marker and an independent indicator of poor prognosis for patients with LSCC.

Identification of LETM1 as a marker of cancer stem-like cells and predictor of poor prognosis in esophageal squamous cell carcinoma

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) is closely related to the occurrence and development of malignant tumors. This study discusses the expression of LETM1 in esophageal squamous cell carcinoma (ESCC) and its association with cancer stem-like cells (CSC). We used immunohistochemistry in 166 ESCC tissue samples, as well as Western blot and immunofluorescent methods in ESCC cell lines, to study the role of LETM1 and its association with CSC in ESCC. The expression of LETM1 was significantly higher in ESCC, and it was closely related to the primary tumor stage and clinical stage. LETM1 expression was significantly associated with lower overall survival and disease-free survival. In addition, the protein expression of LETM1 and CSC markers was higher in TE11 and ECG10 than in other ESCC cell lines. Moreover, the expression of LETM1 positively correlated with LSD1, CD44, and OCT4. Immunofluorescence revealed that LETM1 costained with CD44 and OCT4 in ECG10. The expression of LETM1 was associated with not only HIF-1α but also higher microvessel density and tumor-associated macrophage infiltration. Furthermore, LETM1 significantly correlated with cyclinD1 and pAkt. High expression of LETM1 indicates poor prognosis and may be a potential CSC marker in ESCC. Moreover, LETM1 may be a novel therapeutic target for the treatment of ESCC.

B7H4 is associated with stemness and cancer progression in esophageal squamous cell carcinoma

B7H4 is overexpressed in human cancers and often correlates with poor clinical outcome. There is a lack of data on the role of B7H4 as a cancer stem cell (CSC) regulator in esophageal squamous cell carcinoma (ESCC) and its expression levels compared to other stemness genes in ESCC. In this study, we have assessed the expression of B7H4 and cancer stemness proteins in 156 paraffin-embedded ESCC tissue samples using immunohistochemistry as well as in ESCC cell lines using Western blotting and immunofluorescence imaging. The correlation of B7H4 expression with clinicopathological parameters, cell cycle regulating genes, and PI3K/Akt/NF-κB signaling genes was investigated. The expression of B7H4 in ESCC tissue was correlated with the primary tumor (pT) stage, stromal activity, and the expression of CD68 and HIF-1α. However, B7H4 expression was negatively associated with CD8+ T cell infiltration in ESCC tissues. Moreover, B7H4 was found to be strongly linked to prognostic factors leading to poor clinical outcome. B7H4-expressing cancer cells also expressed known cancer stemness proteins (Sox9, LSD1, Oct4, and LGR5). Moreover, B7H4, Sox9, LSD1, Oct4, and LGR5 were highly expressed in more poorly differentiated ESCC cell lines. Notably, B7H4 expression was positively associated with the expression of cell cycle regulators such as cyclin D1, p27, and PI3K/Akt/NFκB signaling proteins. B7H4 could be a novel cancer stem cell marker for the prognostic evaluation of ESCC patients as well as a potential therapeutic target against ESCC.

Tenascin-C as a prognostic determinant of colorectal cancer through induction of epithelial-to-mesenchymal transition and proliferation

Although Tenascin-C (TNC) as an extracellular matrix protein involved in various cancers, the mechanisms by which TNC leads to decreased survival time remain to be clarified in CRC. We assessed the expression of TNC and its relationship with cancer associated fibroblasts (CAFs) markers, epithelial-to-mesenchymal transition (EMT) and cell cycle markers in 100 paraffin-embedded CRC tissue samples using immunohistochemistry. TNC expression was higher in CRC tissue samples than in adjacent non-tumor-tissues (P < .001). In addition, TNC was involved in clinical stage (P = .030), pT stage (P = .049), distant metastasis (P = .004), tumor recurrence (P = .007), and tumor budding (P < .001). TNC play crucial roles in regulating the poor 5-year CRC survival rate by Kaplan-Meier analysis, and was an independent predictor of poor overall survival (P = .007) and disease-free survival (P = .004) in CRC. Moreover, it was postively correlated with CAF (SMA (P < .001) and FSP1 (P = .005)) and cell cycle marker p27 (P = .013) along with EMT (E-cadherin, P = .599; Snail, P < .001; vimentin, P = .012). TNC may promote EMT-like change and proliferation, which lead to poor prognosis for patients with CRC.

GLI1 promotes cancer stemness through intracellular signaling pathway PI3K/Akt/NFκB in colorectal adenocarcinoma

&NA; The role of Hedgehog (HH)/ glioma‐associated oncogene homolog 1 (GLI1) pathway has been implicated in a variety of cancer entities, and the targeted pathway inhibition mediated by GLI1 is of therapeutic relevance. However, its oncogenicity and cross‐talks with other cancer pathways including PI3K/Akt/NF&kgr;B, which modulates the HH/GLI1 signal strength, have rarely been explored in colorectal adenocarcinoma. We assessed the expression of GLI1 and its relationship with other cancer stemness genes, cell cycle markers, epithelial‐mesenchymal transition (EMT), PI3K/Akt/NF&kgr;B signaling pathway genes, and HIF1&agr; in 100 paraffin‐embedded colorectal adenocarcinoma tissue samples using immunohistochemistry. We further addressed the effect of GLI1 on EMT, cell cycle, and its putative interaction with the PI3K/Akt/NF&kgr;B cascade in colorectal adenocarcinoma cell lines. The expression of GLI1 in colorectal adenocarcinoma tissues was found to correlate with the clinical stages, and distant metastasis. Moreover, GLI1 was found to be an independent predictor of poor overall survival and disease‐free survival in colorectal adenocarcinoma. GLI1‐expressing cancer cells also expressed their representative cancer stem‐like cell (CSC) markers (SOX9 and CD133), as well as HIF1&agr;. GLI1 expression was also strongly linked to EMT‐related and PI3K/Akt/NF&kgr;B signaling genes. Downregulation of GLI1 by inhibitor treatment in colorectal adenocarcinoma cell lines resulted in reduced expression of CSC markers, cell clonogenicity, S‐phase subpopulations, as well as the migration and invasion ability. Importantly, Akt inhibitor Perifosine significantly inhibited the expression of pAkt and GLI1 in colorectal adenocarcinoma cells. Combination of GLI1 inhibitor GANT61 and NF&kgr;B p65 inhibitor QZN exhibited much higher inhibition compared to using any of them individually on colorectal adenocarcinoma cells. We suggested that GLI1 may be a novel stem cell marker, and cancer stemness was activated via PI3K/Akt/NF&kgr;B pathway. In addition, co‐targeting GLI1 and PI3K/Akt/NF&kgr;B signaling simultaneously might provide an alternative therapeutic strategy for colorectal adenocarcinoma patients. HighlightsGLI1 is a prognostic determinant in colorectal adenocarcinoma.GLI1 may be a potential cancer stem‐like cells marker in colorectal adenocarcinoma.GLI1 activated cancer stemness probably via the PI3K/Akt/NF&kgr;B signaling.Co‐targeting GLI1 and PI3K/Akt/NF&kgr;B signaling simultaneously might provide an alternative therapeutic strategy for colorectal adenocarcinoma patients.

Gli1 is a potential cancer stem cell marker and predicts poor prognosis in ductal breast carcinoma

Glioma-associated oncogene homolog 1 (Gli1) maintains the cancer stem cell-like characteristics in various tumors. However, its expression in cancer stem cells (CSC) in ductal breast carcinoma has not been well studied. We aimed to characterize Gli1 as a potential CSC marker and investigate its clinical significance in ductal breast carcinoma. Immunohistochemical staining was used to study the relationship of Gli1 to clinicopathologic features, cell cycle regulation-related genes, and CSC markers. Gli1 was expressed to a greater extent in ductal breast carcinoma than in normal breast tissues (P=.002). Its expression was significantly correlated with tumor grade (P=.044), pT stage (P=.017), and molecular subtype (P=.008). Expression was associated, not only with the expression of HIF-1α (P<.001), but also with greater microvessel density (MVD; P=.012). Kaplan-Meier survival analysis revealed that Gli1 was significantly associated with lower overall survival (OS; P=.02). Univariate Cox regression analysis confirmed that Gli1 was a poor prognostic factor for OS (P=.037) and was associated with the expression of the cell cycle-related genes cyclin D1 (P=.011), p21 (P=.009), and pAkt-Thr308 (P=.038). Moreover, Gli1 expression correlated significantly with the expression of two CSC markers, Sox2 (P=.01) and LSD1 (P=.01). Gli1 could be a stem cell marker and an indicator of poor prognosis in patients with ductal breast carcinoma.