B7-H3 contributes to the metastatic capacity of melanoma cells by modulation of known metastasis-associated genes.

B7-H3, an immunoregulatory protein, is known to play a role in tumor progression. In many cancer types, observed correlations between high B7-H3 expression and poor prognosis have been attributed to involvement in antitumor immunity. However, here we demonstrate a nonimmunological alternative function of B7-H3 in cancer metastasis. Since advanced malignant melanoma is a disease with a poor survival rate and a broad pattern of metastasis, we used this disease as a model in our studies. We found that shRNA silencing of B7-H3 reduced the in vitro migratory potential and matrigel invasiveness of MDA-MB-435 and FEMX-I melanoma cells. In an experimental metastasis model in vivo, B7-H3 silencing of MDA-MB-435 cells resulted in reduced metastatic capacity and significantly increased the median symptom-free survival of nude mice (147 vs. 65 days, p < 0.001) and rats (53 vs. 42 days, p = 0.025) injected with MDA-MB-435 cells. Furthermore, a smaller fraction of mice had microscopically detectable metastases compared to control animals, and the pattern of metastases was slightly different between the two groups but with the brain as the predominant organ. Immunohistochemistry on samples from two melanoma patients showed strong B7-H3 staining in both a primary tumor and metastases. Notably, the metastasis-associated proteins, matrix metalloproteinase (MMP)-2, signal transducer and activator of transcription 3 (Stat3), and the level of secreted interleukin-8 (IL-8) were reduced in the B7-H3 knock-down cell variants, whereas tissue inhibitor of metalloproteinase (TIMP)-1 and-2 levels were increased. Taken together, our findings indicate a novel role for B7-H3 in the regulation of the metastatic capacity of melanoma cells and it might be a potential therapeutic target for anti-metastasis therapy.

Dipeptidyl peptidase IV expression in cancer and stromal cells of human esophageal squamous cell carcinomas, adenocarcinomas and squamous cell carcinoma cell lines.

Dipeptidyl peptidase IV (DPPIV) is a transmembrane serine protease which is involved in the process of tumor invasion and development of metastases in human cancers. The aim of this study was to investigate the expression of DPPIV in cancer and stromal cells of both esophageal adenocarcinoma and squamous cell carcinoma (SCC). Tissue material from 159 patients was analyzed using immunohistochemistry. Western blotting was performed on cell lines and fresh frozen tissue sections. Results were compared with clinicopathological features. Evaluation of the immunohistochemical findings revealed significant differences between DPPIV expression in carcinoma cells and stromal cells, depending on the histological tumor type. A significantly higher level of DPPIV was found in adenocarcinomas compared to SCCs while no DPPIV was detected in normal esophageal epithelium. Overexpression of DPPIV in patients with adenocarcinoma was additionally associated with distant metastases. Thus, differences of DPPIV level in esophageal carcinomas compared with normal epithelium showed that esophageal malignancies were associated with an increased amount of cell surface-bound DPPIV. Radiotherapy in patients had no impact on DPPIV expression in analyzed tissue samples. There was no correlation between DPPIV expression in cancer or stromal cells and survival of the patients.

Seprase, dipeptidyl peptidase IV and urokinase-type plasminogen activator expression in dysplasia and invasive squamous cell carcinoma of the esophagus. A study of 229 cases from Anyang Tumor Hospital, Henan Province, China.

OBJECTIVE: Seprase, dipeptidyl peptidase IV (DPPIV) and urokinase-type plasminogen activator (uPA) play a crucial role in the degradation of the extracellular matrix and in the progression of various human tumors. However, their pathophysiologic significance in esophageal carcinoma has not yet been fully elucidated. METHODS: The expression of seprase, DPPIV and uPA in esophageal dysplasia, squamous cell carcinoma (SCC) and normal epithelium was examined by immunohistochemistry. RESULTS: Seprase, DPPIV and uPA immunoreactivity was found in dysplastic and cancer cells as well as in stromal cells adjacent to dysplasia and cancer sites, but not in normal epithelium. We found a significant association between uPA expression and sex, tumor size and histological classification in carcinomas. High expression of DPPIV in cancer cells correlated with longer survival of the patients. No significant associations between seprase and clinicopathological features either in dysplasia or in carcinomas were found. Finally, we demonstrated higher levels of seprase, DPPIV and uPA in SCC cell lines than in normal esophageal epithelial cell lines. CONCLUSIONS: Our results showed that seprase, DPPIV and uPA are expressed in both premalignant and malignant forms of SCC, but are lacking in normal esophageal epithelia, suggesting that they are involved in SCC neoplastic progression.

FAP-alpha and uPA show different expression patterns in premalignant and malignant esophageal lesions.

Fibroblast activation protein-alpha (FAP-alpha) and urokinase-type plasminogen activator (uPA) are serine proteases involved in cancer invasion and metastasis. The authors examined FAP-alpha and uPA expression in premalignant and malignant stages of esophageal adenocarcinoma by immunohistochemistry. Additionally, Western blotting was performed on fresh-frozen tissue samples. FAP-alpha and uPA were detected in metaplastic, dysplastic, and carcinoma cells, as well as in adjacent stroma. Stromal FAP-alpha expression was associated with depth of tumor invasion, while stromal uPA expression correlated with lymph node metastases in adenocarcinomas. Stromal uPA expression in cells with premalignant changes correlated with histological grading. Immunoblotting showed higher protease expression in carcinoma tissues than in normal esophageal epithelium. These results suggest that FAP-alpha and uPA expression in metaplastic, dysplastic, and esophageal cancer tissue is associated with neoplastic progression of esophageal lesions.

Tumor Budding, EMT and Cancer Stem Cells in T1-2/N0 Oral Squamous Cell Carcinomas.

BACKGROUND: Early oral carcinomas have a high recurrence rate despite surgery with clear margins. In an attempt to classify the risk of recurrence of oral squamous cell carcinomas, we explored the significance of tumor budding, epithelial-mesenchymal transition (EMT) and certain cancer stem cell markers (CSC). MATERIALS AND METHODS: Tumor budding (single cells or clusters of ≤5 cells in the tumor front, divided into high- and low-budding tumors), EMT and CSC markers were studied in 62 immunohistochemically stained slides of T1/2N0M0 oral squamous cell carcinomas. Tissues and records of follow-up were obtained from the Oslo University Hospital, Norway. Tumor budding, EMT and CSC markers were scored and analyzed. RESULTS: The only significant prognostic marker was tumor budding (p=0.043). Expression of the EMT marker E-cadherin was lost from the invasive front and tended to be a prognostic factor (p=0.17), and up-regulation of vimentin in tumor cells in the invasive front was found; this indicates that EMT had occurred. CSC markers were not associated with recurrence rate in the present study. CONCLUSION: A high budding index was related to poor prognosis in patients with oral cancer. Budding was associated with EMT-like changes. CSC factors were detected but reflected differentiation rather than stemness. Scoring of buds in patients with oral cancer may help discriminate invasive tumors prone to relapse, and thus, provide an indication for adjuvant therapy.

PDGFR-α and CD117 Expression Pattern in Esophageal Carcinomas.

BACKGROUND: Despite advanced diagnostics and multimodal treatments, the overall 5-year survival rate for patients with esophageal cancer remains low. In the past, several specific antibodies, including tyrosine kinase inhibitors, targeting different steps of carcinogenesis were investigated. We examined two receptor tyrosine kinases, platelet-derived growth factor receptor (PDGFR-α) and mast/stem cell growth factor receptor (CD117) in esophageal carcinomas. MATERIALS AND METHODS: Tissue samples of 52 Norwegian patients who underwent esophagectomy were examined using immunohistochemistry. RESULTS: PDGFR-α and CD117 expression was observed in cancer cells in all samples of both carcinoma types. A higher PDGFR-α immunoreactivity was detected in the squamous cell carcinoma group (p=0.032). Surprisingly, a higher number of PDGFR-α-positive cells in the analyzed samples for the entire population was associated with longer survival (p=0.05). CONCLUSION: The findings of our study need to be further validated as we examined a low number of patients. Both PDGFR-α and CD117 probably play an important role in the progression of esophageal carcinoma, and they may possibly be targets for biological anticancer therapy in the future.

Nuclear, cytoplasmic, and stromal expression of ZEB1 in squamous and small cell carcinoma of the esophagus.

Zinc finger E-box-binding homeobox 1 (ZEB1) is a transcriptional factor known to repress E-cadherin promoter and thus induce EMT. Expression of ZEB-1 has in numerous cancers been associated with aggressive disease and poor clinical outcome. Our aim was to investigate the expression of ZEB1 in esophageal squamous- and small-cell carcinomas. Immunohistochemical staining was performed on tissue sections obtained from 151 patients with esophageal squamous cell carcinoma (ESCC) and 25 patients with primary small-cell carcinoma of the esophagus (PSCCE). Semi-quantitative analysis, and thus statistical analysis, has been accomplished on the samples. Immunohistochemistry revealed ZEB1 expression in the cytoplasm (64.9% of cases), in nuclei (11.3% of cases) and in tumor stroma (80.1% of cases) of ESCC. In PSCCE only nuclear staining (88.0% of cases) was observed. Weak cytoplasmic expression of ZEB1 in ESCC was associated with longer survival. Immunohistochemical evaluation of ZEB1 cytoplasmic expression in ESCC may have clinical prognostic value, but further studies are needed to fully understand the function as well as potential clinical and therapeutic implications of ZEB1 expression in cancers.

Primary tumor vascularity in esophagus cancer. CD34 and HIF1-α expression correlate with tumor progression.

OBJECTIVE: Hypoxia inducible factor α (HIF1-α) is a key protein regulating the response of a variety of genes and pathways, including angiogenesis, to hypoxic stimuli. High vascularity in various carcinomas correlates with invasion and metastasis. Assessment of primary tumor vascularity and HIF1-α expression in esophageal carcinomas was an objective of this study. METHODS: The vascularity in esophageal carcinomas (n=52) was quantified by Chalkley method on CD34 immunostained sections. HIF1-α expression was examined by immunohistochemistry. The relationships between CD34 Chalkley count, HIF1-α and various clinico-pathological characteristics with clinical outcome were evaluated. RESULTS: High HIF1-α expression in squamous cell carcinoma (SCC) was significantly associated with the T3-4 group (p=0.02). A higher percentage of SCC with high HIF1-α expression compared to its expression in adenocarcinoma (AC) (p=0.005) was observed. In the SCC group, high CD34 Chalkley count and high HIF1-α expression implied a significantly reduced survival (p=0.003 and p=0.001). No such significant association was found in the AC group. CONCLUSIONS: HIF1-α expression is different in two separate tumor microenvironments: SCCs and ACs of the esophagus. This suggests that different mechanisms may be involved in HIF1-α expression- and activity between the two histological types of esophageal carcinoma.

Expression of SSTR2a, but not of SSTRs 1, 3, or 5 in somatotroph adenomas assessed by monoclonal antibodies was reduced by octreotide and correlated with the acute and long-term effects of octreotide.

CONTEXT: Reduced expression of somatostatin receptors (SSTRs) in somatotroph adenomas and their potential down-regulation after medical treatment may explain the unsatisfactory response to octreotide in particular acromegalic patients. The expression of SSTRs other than SSTR2a has not been studied in large, unselected cohorts using novel rabbit monoclonal antibodies. OBJECTIVE: We aimed to determine the expression of SSTRs 1, 2a, 3, and 5 in somatotroph adenomas, to correlate expression with clinical characteristics and the response to octreotide, and to ascertain whether preoperative octreotide treatment affected SSTR expression. DESIGN, SETTING, PATIENTS: The study included 78 adenomas from patients operated on consecutively during 2000 to 2010. After exclusion of 13 patients, immunohistochemical analysis with rabbit monoclonal antibodies against SSTRs 1, 2a, 3, and 5 (clones UMB-7, -1, -5, and -4) was performed on 65 adenomas. INTERVENTION: Twenty-eight patients received preoperative octreotide, and 37 patients were operated on without pretreatment. Twenty-six patients were randomized to direct surgery (n = 13) or to octreotide pretreatment (n = 13). MAIN OUTCOME MEASURE: SSTR expression was evaluated using a 12-grade scoring system. The responses to the octreotide test dose (GH reduction) and to 6 months of octreotide (IGF-I reduction) were measured. RESULTS: The majority of adenomas showed membranous expression of SSTRs 2a and 5. SSTR2a expression was reduced in the pretreated group and correlated with the acute octreotide test results and the effect of octreotide treatment. In a linear regression model with SSTR2a expression as the determinant, the correlation with the acute test response improved after adjustment for medical pretreatment. CONCLUSION: Rabbit monoclonal antibodies are reliable markers of SSTRs in somatotroph adenomas. SSTR2a expression correlated with the response to octreotide and was reduced after octreotide treatment, indicating the need for adjustment when SSTR2a expression is correlated with baseline characteristics. Evaluation of SSTR subtypes may be an important aspect of improving the medical treatment for acromegaly.

Hypoxia-independent downregulation of hypoxia-inducible factor 1 targets by androgen deprivation therapy in prostate cancer.

PURPOSE: We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred. METHODS AND MATERIALS: Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvant ADT was assessed by immunohistochemistry. RESULTS: A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT (P<.001). CONCLUSIONS: AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to tumor regression. HIF1α expression is probably not a useful hypoxia biomarker during ADT in prostate cancer.

KIT protein expression and mutational status of KIT gene in pituitary adenomas.

KIT protein expression and mutational status of KIT gene in different types of tumours have been intensively studied since Imatinib Mesylate, KIT/PDGFRA tyrosine kinase inhibitor became available. However, only one immunohistochemical study on KIT expression in pituitary adenomas has been published. There are currently no reports on mutational status of KIT gene in pituitary adenomas. We have immunohistochemically investigated KIT expression in 252 pituitary adenomas and found cytoplasmic reactivity in 52.4% and membranous reactivity in 8.3% of all adenomas. There was statistically significant difference in KIT expression between clinically non-functioning, growth hormone- and adrenocorticotroph hormone-producing adenomas. The group with membranous expression was dominated by somatotropinomas and clinically non-functioning adenomas. KIT expression in a subset of adenomas was also confirmed by western blot analysis of 48 adenomas. Immunohistochemical KIT expression was correlated with basic clinical data and in a cohort of acromegalic patients with additional data (somatostatin receptor type 2A expression, response to somatostatin analogue treatment and mutational status of gsp oncogene). Exons 9, 11, 13 and 17 of KIT gene were searched for mutations in the tumours with membranous KIT expression and in a minority of tumours with cytoplasmic KIT expression using denaturing high-performance liquid chromatography and in suspected cases sequencing of one or more exons. No mutations in the examined exons were found. Our results may suggest a role of KIT in the pathogenesis of a subset of pituitary adenomas and point out the need for further research to find out if KIT-reactive adenomas could be sensitive to Imatinib Mesylate.

B7-H3 silencing increases paclitaxel sensitivity by abrogating Jak2/Stat3 phosphorylation.

In many types of cancer, the expression of the immunoregulatory protein B7-H3 has been associated with poor prognosis. Previously, we observed a link between B7-H3 and tumor cell migration and invasion, and in present study, we have investigated the role of B7-H3 in chemoresistance in breast cancer. We observed that silencing of B7-H3, via stable short hairpin RNA or transient short interfering RNA transfection, increased the sensitivity of multiple human breast cancer cell lines to paclitaxel as a result of enhanced drug-induced apoptosis. Overexpression of B7-H3 made the cancer cells more resistant to the drug. Next, we investigated the mechanisms behind B7-H3-mediated paclitaxel resistance and found that the level of Stat3 Tyr705 phosphorylation was decreased in B7-H3 knockdown cells along with the expression of its direct downstream targets Mcl-1 and survivin. The phosphorylation of Janus kinase 2 (Jak2), an upstream molecule of Stat3, was also significantly decreased. In contrast, reexpression of B7-H3 in B7-H3 knockdown and low B7-H3 expressing cells increased the phosphorylation of Jak2 and Stat3. In vivo animal experiments showed that B7-H3 knockdown tumors displayed a slower growth rate than the control xenografts. Importantly, paclitaxel treatment showed a strong antitumor activity in the mice with B7-H3 knockdown tumors, but only a marginal effect in the control group. Taken together, our data show that in breast cancer cells, B7-H3 induces paclitaxel resistance, at least partially by interfering with Jak2/Stat3 pathway. These results provide novel insight into the function of B7-H3 and encourage the design and testing of approaches targeting this protein and its partners.

Expression of the epidermal growth factor receptor family in normal and malignant urothelium.

OBJECTIVE: To compare the immunohistochemically assessed expression of the epidermal growth factor receptor (EGFR) family in normal and malignant bladder urothelium, and suggest new hypotheses about their function in the development and progression of transitional cell carcinoma (TCC). PATIENTS AND METHODS: EGFR, ERBB2, ERBB3 and ERBB4 were evaluated immunohistochemically in normal urothelium (NU, 15), primary non-metastasized invasive TCC (NMC, 19) and in primary invasive TCCs with corresponding metastases (MC, 51, both specimens). RESULTS: All NU samples expressed ERBB4, none expressed ERBB2 and two expressed EGFR; all staining was uniform throughout all cell layers. ERBB2 expression increased and ERBB4 decreased from normal samples to carcinomas. There was no difference between NMCs and MCs in ERBB2, ERBB3 and ERBB4, but the NMCs expressed more EGFR than both NU and MC samples. There were no associations with T category, grade or survival. All combinations of expression levels for the four receptors were detected, with no dominant profile. CONCLUSION: We hypothesise that: (i) ERBB4 is important for differentiation in NU; (ii) ERBB2 is up-regulated with carcinogenesis in the urinary bladder but does not discriminate between bladder cancer with or without metastases; (iii) EGFR may be a marker of indolent disease. A current hypothesis, that superficial layers of NU do not express EGFR and thus protect the basal cells from the mitogenic effect of urinary EGF, is challenged.