Metastatic cancer cells from c-erbB-2 negative primary breast cancer maintain the original c-erbB-2/HIF1alpha phenotype

The expression of c-erbB-2 and HIF1alpha proteins is linked with an aggressive tumor phenotype and poor survival in breast cancer. In the present study we investigated whether this ominous effect is a result of c-erbB-2/HIF1alpha expressing clone appearance within the primary tumor, by examining the c-erbB-2/HIF1alpha expression status in the primary tumor, node metastasis and cancer cells invading into the lymphovascular spaces. The metastasizing cancer cell clones, whether migrating to the lymph nodes or entering into the systemic circulation maintained the original c-erbB-2/HIF1alphaphenotype of the primary tumor. Migrating c-erbB-2/HIF1alpha negative tumors do so through activation of alternative biologic pathways and not through positive clone appearance. These results strongly support the concept that targeted therapies against c-erbB-2 or against HIF1alpha can be guided with precision by the primary tumor c-erbB-2/HIF1alpha status without demanding the detection of the metastatic tumor phenotype.     

Prognostic impact of hypoxia-inducible factors 1alpha and 2alpha in colorectal cancer patients: correlation with tumor angiogenesis and cyclooxygenase-2 expression.

PURPOSE: Angiogenesis plays an important role in a multitude of biological processes including those of tumorigenesis and cancer progression. Hypoxia is the prime driving factor for tumor angiogenesis and the family of hypoxia-inducible factors (HIFs) plays a pivotal role in this process. The role of HIF in tumor angiogenesis has been underscored in different carcinomas but yet to be reported for colorectal carcinomas. EXPERIMENTAL DESIGN: In this study, we examined HIF [HIF-1alpha (HIF1) and HIF-2alpha (HIF2)] expression in 87 curatively resected colorectal carcinoma samples, and the results were correlated with clinicopathological factors, microvessel density, cyclooxygenase 2 expression, and patient prognosis. RESULTS: HIF1 (44.8%) was more frequently expressed than HIF2 (29.9%). Most of the clinicopathological factors representing the tumor aggressiveness were significantly correlated with overexpression of HIF2 but not with HIF1 expression. HIF2 expression had direct correlation with microvessel density and cyclooxygenase 2 expression. and, in contrast, HIF1 expression had a weak but significant inverse correlation in T1 and T2 tumors only. HIF2 expression alone and the combined expression of HIF1 and HIF2 had significant impact on patient survival. In the multivariate analysis, however, only the combined expression of HIF1 and HIF2 remained independently significant. CONCLUSIONS: Taken together, our results suggest that HIF2 expression may play an important role in angiogenesis and that the combined expression of HIF1 and HIF2 may play an important role in tumor progression and prognosis of colorectal carcinomas. Therefore, HIF expression could be a useful target for therapeutic intervention.     

Expression of hypoxia-inducible carbonic anhydrase-9 relates to angiogenic pathways and independently to poor outcome in non-small cell lung cancer.

Carbonic anhydrase-9 (CA9), a transmembrane enzyme with an extracellular active site, is involved in the reversible metabolism of the carbon dioxide to carbonic acid. Up-regulation of CA by hypoxia and the hypoxia-inducible factor (HIF) pathway has been recently postulated (Wykoff et al. Cancer Res., 60: 7075-7083, 2000). In the present study we examined the expression of this enzyme in non-small cell lung cancer. Of 107 cases analyzed, 39 (36.4%) had strong membrane/cytoplasmic expression of CA9 and were grouped as positive. The staining was confined around areas of necrosis, and a significant association of CA9 expression with the extent of necrosis was noted (P = 0.004). Nevertheless, 38 of 74 cases with focal or extensive necrosis did not express CA9. CA9 expression was more frequent in the squamous cell histology (P = 0.001) and with advanced T stage (P = 0.009). A significant coexpression of CA9 with platelet-derived endothelial cell growth factor and basic fibroblast growth factor receptor expression was noted. Double staining of CA9 with anti-CD31 monoclonal antibody revealed an overall higher microvessel density in the areas expressing CA9 than in negative areas (P = 0.0005). Thirty-one of 38 CA9-positive cases were positive for HIF1a/HIF2a, but HIF positivity was a more common event (68 of 107) and their patterns of expression were diffuse (not confined in the necrotic areas). A direct association of CA9 expression with epidermal growth factor receptor, c-erbB-2, and MUC1 expression was also noted (P < 0.04). Survival analysis showed that CA9 expression is related to poor prognosis. CA9 expression in tumors with low vascularization defined a prognosis similar to the one of patients with highly angiogenic tumors. Multivariate analysis revealed that CA9 expression is a significant prognostic factor independent of angiogenesis. We conclude that CA9 is an important molecule in non-small cell lung cancer, the up-regulation of which occurs in highly hypoxic/necrotic regions of the tumors. The expression of CA9 is linked to the expression of a constellation of proteins involved in angiogenesis, apoptosis inhibition, and cell-cell adhesion disruption, which explains the strong association of CA9 with poor outcome.     

Targeted inactivation of fh1 causes proliferative renal cyst development and activation of the hypoxia pathway

Germline mutations in the fumarate hydratase (FH) tumor suppressor gene predispose to leiomyomatosis, renal cysts, and renal cell cancer (HLRCC). HLRCC tumors overexpress HIF1alpha and hypoxia pathway genes. We conditionally inactivated mouse Fh1 in the kidney. Fh1 mutants developed multiple clonal renal cysts that overexpressed Hif1alpha and Hif2alpha. Hif targets, such as Glut1 and Vegf, were upregulated. We found that Fh1-deficient murine embryonic stem cells and renal carcinomas from HLRCC showed similar overexpression of HIF and hypoxia pathway components to the mouse cysts. Our data have shown in vivo that pseudohypoxic drive, resulting from HIF1alpha (and HIF2alpha) overexpression, is a direct consequence of Fh1 inactivation. Our mouse may be useful for testing therapeutic interventions that target angiogenesis and HIF-prolyl hydroxylation.     

Significance of immunohistochemically detected c-erbB-2 protein expression in stage III breast cancer, with reference to nuclear deformity, DNA content and prognosis.

The significance of c-erbB-2 protein expression was immunohistochemically investigated in 32 patients with stage III breast cancer with regard to area, shape factor and DNA content of cancer cell nuclei. Thirty-one percent of the tumors showed c-erbB-2-positive cell membrane staining (10 out of 32 cases), and the cancer cells of the c-erbB-2-positive tumors had larger and more irregular nuclei with an increased DNA content. All patients with c-erbB-2 protein-positive tumors died of the disease within six years of their mastectomy. It can be concluded that positive immunohistochemical staining of c-erbB-2 protein indicates an aggressive biological behavior of the cancer cells as well as a poor prognosis in patients with stage III breast cancer.     

Hypoxia-inducible factor (HIF1A and HIF2A), angiogenesis,and chemoradiotherapy outcome of squamous cell head-and-neck cancer

PURPOSE: Hypoxia-inducible factors HIF1alpha and HIF2alpha (HIFalphas) regulate the expression of a variety of genes encoding proteins related to angiogenesis and to anaerobic metabolism of cells exposed to hypoxic stress. Their putative role as markers of clinically relevant hypoxia and, therefore, as predictors of response to chemoradiotherapy is herein examined. PATIENTS AND METHODS: Using immunohistochemistry, we assessed the expression of HIFalphas in normal head-neck mucosa and in 75 cancer specimens from patients with locally advanced squamous cell head-and-neck cancer (SCHNC), treated with concurrent carboplatin chemoradiotherapy. RESULTS: Head-and-neck mucosa from normal individuals did not show any HIF1alpha or HIF2alpha reactivity. SCHNC showed a varying expression of HIFalphas ranging through negative reactivity, to weak or focally strong cytoplasmic reactivity, or to strong diffuse cytoplasmic/nuclear reactivity. Fifty-two percent and 33% of cancer samples showed the latter expression pattern for HIF1alpha and HIF2alpha, respectively, and were considered to bear "high" HIF reactivity. Bone/cartilage involvement was more frequent in tumors with high HIF1alpha expression (p = 0.05). HIF1alpha and HIF2alpha overexpression were significantly associated with high microvessel density (p = 0.002 and 0.02, respectively) and with VEGF expression (p = 0.01 and 0.005, respectively). HIF1alpha was related to high thymidine phosphorylase expression (p = 0.03), whereas VEGF/KDR-activated tumor vasculature was significantly more frequent in HIF2alpha-overexpressing tumors (p = 0.02). High HIF1alpha and HIF2alpha were associated with incomplete response to chemoradiation (p = 0.007 and p = 0.02, respectively). In univariate analysis, high HIF1alpha and HIF2alpha expression were significantly associated with poor local relapse-free survival (p = 0.003 and 0.003, respectively) and with poor overall survival (p = 0.05 and 0.001, respectively). In multivariate models, HIF2alpha expression was an independent prognostic factor. In biopsies performed after the delivery of 20 Gy of radiotherapy, upregulation of HIFalphas was noted in some cases. CONCLUSIONS: It is concluded that the overexpression of HIFalphas in SCHNC is related to locally aggressive behavior, to intensification of angiogenesis, and to an important resistance to carboplatin chemoradiotherapy.     

Prognostic significance of c-erbB-2 and estrogen receptor status in human breast cancer.

Using the 21N polyclonal antibody, we immunohistochemically stained 314 primary breast carcinomas to identify those tumors overexpressing the c-erbB-2 oncoprotein and to ascertain the prognostic significance of this expression on disease-free and overall survival. Positive membrane staining was present in 52 (17%) of these carcinomas of which 7 (13%) were ductal carcinomas in situ. There was no significant relationship between c-erbB-2 positivity and (a) age at diagnosis, (b) menopausal status, (c) tumor size, (d) lymph node status, (e) estrogen receptor status, or (f) whether or not the patient had disseminated disease outside the axillary fields. However, c-erbB-2-positive tumors were significantly associated with poorer grade (P = 0.02). Patients who were positive for this oncoprotein had a shorter disease-free survival (P = 0.002) and reduced overall survival (P = 0.0001). Overexpression of this oncoprotein was predictive of a worse prognosis in lymph node-positive disease (P = 0.003) and in patients presenting with grade II tumors (P = 0.001). Stratifying the patients on the basis of estrogen receptor status suggested that c-erbB-2+/estrogen receptor-negative status was predictive of a poorer prognosis when compared with the other subgroups (P less than 0.001). Primary and recurrent tumor tissues were available from 42 of the 314 patients. Identical patterns of c-erbB-2 expression occurred in 95% of cases, arguing against a direct role for c-erbB-2 expression in the process of tumor dissemination. The high incidence of staining in ductal carcinomas in situ suggests that expression of this oncoprotein is an early event in tumorigenesis. Finally, multivariate analysis indicated that the c-erbB-2 oncoprotein was an independent prognostic indicator for overall survival in breast carcinoma patients.     

Endogenous markers of hypoxia/anaerobic metabolism and anemia in primary colorectal cancer

Anemia has been implicated in the decreased oxygen tension noted within the tumor environment. In a series of 79 colorectal adenocarcinomas we investigated the role of anemia in activating molecular pathways regulated by hypoxia. Preoperative Hb levels were correlated with the immunohistochemical expression of HIF1alpha and HIF2alpha, LDH5, GLUT1, VEGF, DEC1 and BNIP3, and with angiogenesis and the cancer cell proliferation index. Upregulation of HIF1alpha and HIF2alpha proteins, found in 43% and 44.3% of cases, respectively, was not related to anemia (Hb < 10 g%). This is in agreement with other studies suggesting that HIF activation occurs for various reasons, such as poor or irregular vascularity, or oncogene activation. Nevertheless, low Hb levels (<10 g%) were linked to activated anaerobic metabolism (LDH5 overexpression) in a subset of tumors not expressing HIF1alpha (P < 0.01). Overexpression of HIFs, whether linked to anemia or not, was associated with a number of factors related to tumor aggressiveness (assessed as local invasion and nodal metastasis), anaerobic metabolism and intratumoral acidosis (LDH5, GLUT1; increased glucose metabolism to lactate), activation of genes related to necrosis (BNIP3) and angiogenesis (VEGF). Expression of BNIP3 emerged as the strongest independent factor related to transmural invasion and metastasis to lymph nodes. Identification of specific patterns of the hypoxia molecular cascade activated in cancer cells might help in developing specific therapeutic policies.     

c-erbB-2 and the “triple-state” in early breast carcinomas

Although c-erbB-2 expression is, in general terms, an ominous prognostic indicator in breast carcinomas, there are suggestions that lack of this oncogene, when combined with analogous lack of estrogen (ER negative) and progesterone receptors (PgR negative)—“triple-negative phenotype”, is linked with an equally poor prognosis. We investigated this hypothesis in a series of early ductal breast carcinomas. A total of 116 specimens with early breast cancer, defined as tumors of ≤2 cm in size and clinically negative axilla, were studied immunohistochemically for ER, PgR, and c-erbB-2 expression. The median follow-up was 131 months (range 62–245 months). ER positive tumors had a favorable clinical course, compared to ER negative neoplasms, but only for the first 10 years of follow-up (P = 0.04). Prognosis was poorer for the PgR negative cases, relative to PgR positive tumors (P = 0.005), but this stood true for the entire investigation period. Triple-negative breast carcinomas had a poor prognosis, while triple-positive tumors had a favorable outcome. However, if triple-positive and triple-negative cases were excluded from the original sample, the remaining c-erbB-2 positive cases were connected with poor prognosis, relative to the remaining c-erbB-2 negative tumors. c-erbB-2 oncogene has a complex biological role in early breast carcinomas for its expression characterizes subgroups of patients with both favorable (triple-positive phenotype) and unfavorable prognosis (c-erb-B2 positive cases after excluding triple-positive and triple-negative tumors)—a phenomenon presumably due to activation of different biological pathways. Elucidation of these pathways may determine subgroups of patients with tumors requiring different targeted agents.     

HIF1A可作为乳腺癌的预后标志物且与免疫细胞浸润相关

目的：借助多种癌症生物信息数据库研究乳腺癌组织中缺氧诱导因子1亚基α(HIF1A)的表达水平及其与乳腺癌患者预后及肿瘤免疫细胞浸润的关系。方法：利用Oncomine、人类蛋白质图谱、基因表达谱交互式分析（GEPIA）及TCGA数据库分析HIF1A基因在乳腺癌组织中的表达及其与患者预后、临床病理特征的关系,并在中国人乳腺癌组织标本（选用2011年1月至2015年12月中国武汉大学人民医院手术切除的93例乳腺癌组织和14例良性乳腺疾病组织）中进行验证。对HIF1A高低表达组间的差异基因进行基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析,用Cibersort R软件评估HIF1A高低表达样本中免疫细胞浸润丰度差异。结果：生物信息数据显示,HIF1A在乳腺癌组织中高表达,预示着患者DFS预后更好（P<0.05）。HIF1A的表达与雌激素受体（ERP）、孕激素受体（PR）和人表皮生长因子受体2(HER2)表达相关（均P<0.05）。GO生物功能及KEGG通路富集分析结果提示,HIF1A可能参与肿瘤免疫调节等生物活动。使用Cibersort分析结果显示,HIF1A与肿瘤...     

c-myc, c-erbB-2, and Ki-67 expression in normal breast tissue and in invasive and noninvasive breast carcinoma.

c-myc, c-erbB-2, and Ki-67 expression was examined by immunohistochemistry in 11 normal breast tissues and 42 invasive and 14 noninvasive breast carcinomas. The c-myc product was detected in all breast carcinoma specimens and in 7 of 11 normal breast tissues. Invasive tumors stained more frequently with the anti-myc monoclonal antibody than did noninvasive tumors, while the level of expression in normal breast tissue was much less than that in breast cancer. Membrane staining of the c-erbB-2 protein was demonstrated in 29% (4 of 14) of noninvasive ductal carcinomas and in 45% (19 of 42) of invasive breast carcinomas. None of the 11 normal breast tissue samples was positive. The mean value of Ki-67-positive cells was 0.91 +/- 0.31% for normal breast tissue, 4.57 +/- 1.36% for noninvasive ductal carcinoma, and 12.76 +/- 2.18% for invasive breast cancer. In 42 invasive breast carcinomas, the expression of c-myc, c-erbB-2, and Ki-67 proliferation marker were compared with lymph node status, estrogen receptor status, progesterone receptor status, and age of patients at diagnosis. c-erbB-2 overexpression and Ki-67 overexpression were identified as the only factors associated with lymph node status. We concluded that they might be additional prognostic factors for breast carcinoma.     

Phase I Trial of Uracil–Ftorafur, Leucovorin, and Etoposide: An Active All-oral Regimen for Metastatic Breast Cancer

To investigate the relation between necrosis and hypoxia in breast cancer we examined the expression of hypoxia-associated markers HIF1, CA IX and GLUT1 by immunohistochemistry in 97 invasive ductal carcinomas. This selected series comprised 48 tumors with extensive necrosis and 49 control tumors without necrosis. Over 90% of necrotic and 30% of non-necrotic tumors expressed at least one hypoxia marker. We also observed expression of hypoxia associated markers in tumor stroma. Examination of primary human breast fibroblasts in vitro confirmed that CA IX mRNA and protein can be induced by hypoxia. Survival analysis of 53 cases found that the subset of tumors with stromal hypoxia exhibit better prognosis (p = 0.027). Our results indicate that necrosis is often accompanied by hypoxia but that hypoxia without necrosis may also be a frequent occurrence. The use of several hypoxia markers may identify a continuum of hypoxia in tumors, which can be sub-classified by different co-expression patterns. We conclude that stromal and epithelial hypoxia may have different biological backgrounds and that stromal hypoxia may affect survival.     

p53 and c-erbB-2 but not bcl-2 are predictive of metastasis-free survival in breast cancer patients receiving post-mastectomy adjuvant radiotherapy in Taiwan

BACKGROUND: Patients with breast cancer often receive radiotherapy after mastectomy if they are at a high risk of local recurrence, but the prognosis varies among patients. We conducted a study to evaluate p53, bcl-2 and c-erbB-2 as predictors of prognosis in breast cancer patients receiving post-mastectomy radiotherapy, which has not been well defined in the Taiwanese population. METHODS: We recruited 74 consecutive patients with primary operable breast cancer who were treated with mastectomy followed by locoregional radiotherapy and studied the presence of p53, bcl-2 and c-erbB-2 expressions in tumor tissues by immunohistochemical staining. Associations between the protein expressions and clinical outcomes, including local recurrence-free survival (LRFS), metastasis-free survival (MFS) and overall survival (OS), were evaluated. RESULTS: The median follow-up time was 55 months. Expressions of p53, bcl-2 and c-erbB-2 were observed in 14 (19%), 28 (38%) and 39 (53%) patients, respectively. Both p53 and c-erbB-2 were significant predictors of MFS. The 5-year MFS for p53-negative and p53-positive tumors were 61.2 and 35.7% (P = 0.01) and 5-year MFS for c-erbB-2-negative and c-erbB-2-positive tumors were 71.3 and 42.4% (P = 0.01). Whereas expression of bcl-2 protein is associated with favorable clinicopathological features, it was not related to LRFS, MFS or OS. Multivariate analyses confirmed c-erbB-2 and p53 expressions as predictors of MFS independent of tumor size, histological grading and lymph node involvement. CONCLUSION: Expressions of p53 and c-erbB-2 are independent predictors of MFS in this Taiwanese population. Further research should be conducted on their application in the treatment and follow-up of patients.     

Small interfering RNA expression vector targeting hypoxia-inducible factor 1 alpha inhibits tumor growth in hepatobiliary and pancreatic cancers

Hepatobiliary and pancreatic carcinomas are hypovascular tumors that can proliferate under hypoxic conditions. Recent reports have demonstrated that hypoxia-inducible factor 1 alpha (HIF1alpha) plays an important role in the survival of these cancers. Given these findings, the inhibition of the HIF1alpha pathway might prove to be a powerful tool in the treatment of these cancers. To inhibit HIF1alpha expression, we used small interference RNA (siRNA) expression vectors in this study. The transient transfection of siRNA expression vectors significantly reduced both HIF1alpha mRNA levels (13% of control) and protein levels (41% of control) and significantly inhibited the growth of cancer cell lines (P<0.05). VEGF, Glut1, and aldorase A expressions were also significantly reduced by transfection with these vectors (P<0.05), and we found that these vectors induced apoptosis but not cell cycle arrest. In a subcutaneous tumor model using nude mice, transfected MIA PaCa-2 cells, stably expressing siRNAs, barely formed tumors compared to control (P<0.05). This study thus demonstrates the usefulness of siRNA expression vector in targeting HIF1alpha and points to a potential clinical role in the treatment of pancreatic and hepatobiliary carcinomas.