Hypoxia-induced factor-1 alpha upregulates vascular endothelial growth factor C to promote lymphangiogenesis and angiogenesis in breast cancer patients

Hypoxia-induced factor-1 alpha （HIF-1α） affects many effector molecules and regulates tumor lymphangio- genesis and angiogenesis during hypoxia. The aim of this study was to investigate the role of HIF-1α in the regu- lation of vascular endothelial growth factor C （VEGF-C） expression and its effect on lymphangiogenesis and an- giogenesis in breast cancer. Lymphatic vessel density （LVD）, microvessel density （MVD） and the expressions of HIF-1α and VEGF-C proteins were evaluated by immunohistochemistry in 75 breast cancer samples. There was a significant correlation between HIF-1α and VEGF-C （P = 0.014, r = 0.273, Spearman＇s coefficient of correlation）. HIF-1α and VEGF-C overexpression was significantly correlated with higher LVD （P = 0.003 and P = 0.017, re- spectively）, regional lymph nodal involvement （P = 0.002 and P = 0.004, respectively） and advanced tumor, node, metastasis （TNM） classification （P = 0.001 and P = 0.01, respectively）. Higher MVD was observed in the group expressing higher levels of HIF-1α and VEGF-C （P = 0.033 and P = 0.037, respectively）. Univariate analysis showed shorter survival time in patients expressing higher levels of HIF-1α and VEGF-C. HIF-1α was also found to be an independent prognostic factor of overall survival in multivariate analysis. The results suggest that HIF-1α may affect VEGF-C expression, thus acting as a crucial regulator of lymphangiogenesis and angiogenesis in breast cancer. This study highlights promising potential of HIF- 1α as a therapeutic target against tumor lymph node me- tastasis.     

Hypoxia-inducible factor-1alpha expression in experimental cirrhosis: correlation with vascular endothelial growth factor expression and angiogenesis

Angiogenesis progresses together with fibrogenesis during chronic liver injury. Hypoxia-inducible factor-1alpha (HIF-1alpha), a master regulator of homeostasis, plays a pivotal role in hypoxia-induced angiogenesis through its regulation of vascular endothelial growth factor (VEGF). The association between hypoxia, angiogenesis and VEGF expression has been demonstrated in experimental cirrhosis. However, expression of HIF-1alpha has yet to be reported. The aim of this study was to investigate the significance of HIF-1alpha expression during experimental liver fibrosis and the relationships between HIF-1alpha expression, VEGF expression and angiogenesis. Cirrhosis was induced in male Wistar rats by intraperitoneal administration of diethyl nitrosamine (DEN) (100 mg/kg, once a week). The serial sections from liver tissues were stained with anti-HIF-1alpha, anti-VEGF and anti-CD34 antibodies before being measured by light microscopy. Our results showed that HIF-1alpha expression gradually increases according to the severity of fibrosis (p<0.01). Moreover, its expression was found to be correlated with angiogenesis (r=0.916) and VEGF expression (r=0.969). The present study demonstrates that HIF-1alpha might have a role in the development of angiogenesis via regulation of VEGF during experimental liver fibrogenesis and suggests that this factor could be a potential target in the manipulation of angiogenesis in chronic inflammatory diseases of the liver.     

Inactivation of the PTEN gene protein product is associated with the invasiveness and metastasis, but not angiogenesis, of breast cancer

PTEN is a novel tumor-suppressor gene located on chromosomal band 10q23. Loss of PTEN function has been implicated in the progression of several types of cancer, but the correlation between loss of PTEN expression and advanced carcinomas is not well established. The capacity for angiogenesis of a tumor is known to play a very important role in growth and metastasis, and there have been reports that PTEN relates to angiogenesis. In the present study, formalin-fixed and paraffin embedded tissues from 101 patients with breast carcinomas, including 88 cases of invasive ductal carcinomas and 13 cases of ductal carcinoma in situ (DCIS), were evaluated by immunohistochemical methods for the expression of PTEN and vascular endothelial growth factor (VEGF), as well as microvessel density (MVD). The results were compared with the clinicopathologic parameters. There was no loss of PTEN expression in any of the cases of DCIS, but 28 (32%) of the 88 invasive cases did not express PTEN. Loss of PTEN expression was associated with lymph node metastasis ( P  = 0.03), but did not correlate with tumor size, tumor grade, MVD or recurrence. VEGF expression significantly correlated with lymph node metastasis in invasive ductal carcinoma ( P  = 0.01). There was no correlation between the expression of PTEN and that of VEGF ( P  = 0.63). The present study suggests that loss of PTEN expression is common and correlates with tumor progression and lymph node metastasis in breast carcinoma. The relationship between loss of PTEN and progression of breast cancer may not be explained by modulation of angiogenesis.     

Cutaneous breast cancer deposits show distinct growth patterns with different degrees of angiogenesis,hypoxia and fibrin deposition

AIMS: We postulated that skin metastases and cutaneous local recurrences from breast adenocarcinoma show different growth patterns with distinct angiogenic profiles. METHODS AND RESULTS: Fifty-one surgically resected dermal breast cancer deposits were evaluated for growth pattern, E-cadherin expression, presence of necrosis and a fibrotic focus, fibrin deposition, carbonic anhydrase IX expression (CA IX), microvessel density, endothelial cell proliferation and blood vessel immaturity. Growth patterns were infiltrative, with carcinoma cells infiltrating the dermis without significant disturbance of the pre-existing architecture, expansive, meaning that a nodule of carcinoma cells and desmoplastic tissue pushed aside the pre-existing dermal structures, or mixed. All lobular carcinomas showed an infiltrative growth and lacked membranous E-cadherin expression. Different growth patterns in the ductal carcinomas were not correlated with differences in E-cadherin expression. The presence of necrosis and/or a fibrotic focus and the expression of the hypoxia marker CA IX were significantly associated with an expansive growth. Fibrin was present in all expansive deposits and less frequently in the other growth patterns. There was a positive association between fibrin deposition, CA IX expression and microvessel density. The latter was significantly higher in the expansive and mixed growth patterns than in the infiltrative pattern. Endothelial cell proliferation was highest in the expansive growth pattern and was positively correlated with the presence of a fibrotic focus and with fibrin deposition. The maximum percentage of immature blood vessels was higher in the expansive and mixed growth patterns than in the infiltrative one. CONCLUSION: The recognition of different subgroups of cutaneous breast cancer deposits with different degrees of hypoxia-driven angiogenesis may have important implications for the usefulness of anti-angiogenic therapy.     

PlGF expression in pre‐invasive and invasive lesions of uterine cervix is associated with angiogenesis and lymphangiogenesis

Most vascular endothelial growth factors (VEGF) have been shown to be associated with lymphangiogenesis and angiogenesis in various cancers. However, whether placental growth factor (PlGF), a rarely mentioned VEGF member, is involved in the pathogenesis of uterine cervical lesions remains unclear. To address this issue, we examined the relationship between PlGF expression and clinicopathologic variables in patients with pre‐invasive and invasive lesions of uterine cervix. Sixty‐two cervical specimens were immunostained with PlGF polyclonal antibody to define PlGF expression, and monoclonal antibodies D2‐40 and CD34 to evaluate the lymphatic vessel density (LVD) and blood vessel density (BVD) of the lesions. PlGF mRNA level was detected by RT‐PCR in all lesions from fresh tissues. We found that the levels of PlGF protein and mRNA expression were related to clinical stages (p < 0.05), but not to other clinicopathologic variables. No significant difference in PlGF expression was observed between squamous carcinoma and adenocarcinoma. Increased LVD and BVD were all associated with advanced stages (p < 0.001). Although LVD was strongly correlated with BVD, only high LVD was associated with pelvic lymphatic metastasis. Moreover, the level of PlGF expression was associated with both BVD(r = 0.715, p < 0.001) and LVD(r = 0.321, p < 0.05). Together, our study suggests that PlGF may participate in both tumor‐associated angiogenesis and lymphangiogenesis of cervical carcinogenesis.     

Angiogenesis in invasive breast carcinoma: is it associated with parameters of prognostic significance?

Recent experimental and clinical studies suggest that tumour-induced angiogenesis may be an important step in the evolution of malignant tumours, and may be related to prognosis. In our study we examined 42 cases of breast carcinoma (mean age: 56.76 ± 13.5), 21 with lymph node metastases and 21 without. Angiogenesis was evaluated after immunohistochemical staining of tumour vessels, using polyclonal antibody to factor VIII related antigen (VIIIR-Ag) and counting of the three most active areas of neovascularization. In the same manner we counted the microvessels in lymph node metastases. The mean vessel count of node-negative cases (51.16 ± 19.32) did not differ significantly from node-positive cases (45.66 ± 17.44). In contrast patients younger than 50 years had much higher mean vessel counts (54.04 ± 16.47) than did patients older than 70 years (38.03 ± 16.73) producing a P value of ≤0.05. No association was found between tumour size and mean vessel count, nor was there any significant difference between grade I (45.94 ± 16.54), grade II (53.13 ± 23.22) and grade III tumours (51.71 ± 20.64). When we compared the mean vessel count of primary tumours with those of node metastases, we found much lower counts in the latter ( P ≤0.01). The differences in our results from previous studies, probably reflect the heterogeneity which exists between different tumours in their ability to induce angiogenesis. Additionally, there is some evidence in our study that angiogenesis is possibly related to patient age and probably depends on differences in the tumour stroma.     

乳腺癌血管生成及组织蛋白酶D表达与转移的关系

目的：探讨乳腺癌组织微血管密度（ｍｉｃｒｏｖｅｓｅｌｄｅｎｓｉｔｙ，ＭＶＤ）及组织蛋白酶Ｄ（ｃａｔｈｅｐｓｉｎＤ，ＣＤ）表达与转移的关系。方法：应用免疫组织化学方法对乳腺癌组织的ＭＶＤ及ＣＤ进行定量和半定量研究。结果：乳腺癌组织ＭＶＤ及ＣＤ表达与淋巴结转移密切相关，腋下淋巴结转移组的ＭＶＤ（１３１．８±３９．８）明显高于腋下淋巴结阴性乳腺癌（ｎｏｄｅｎｅｇａｔｉｖｅｂｒｅａｓｔｃａｎｃｅｒ，ＮＮＢＣ）组的ＭＶＤ（７８．１±２７．１）（Ｐ＜０．００１）；腋下淋巴结转移组ＣＤ高表达者（３３／３６，９１．７％）明显多于ＮＮＢＣ组（１６／３６，４４．４％）（Ｐ＜０．０１）。乳腺癌组织ＭＶＤ与ＣＤ表达关系密切；远处转移及早期死亡者ＭＶＤ较高且ＣＤ表达极强。结论：乳腺癌组织ＭＶＤ的增加及ＣＤ高表达可能协同促进肿瘤转移。富于微血管及ＣＤ高表达的乳腺癌应作为远处转移及早期死亡的高危患者加以重视。     

Decreased HCRP1 expression is associated with poor prognosis in breast cancer patients

Background: Downregulation of hepatocellular carcinoma related protein 1 (HCRP1) has been reported to be associated with a poor prognosis in a variety of malignant tumors. The purpose of this study was to assess HCRP1 expression in breast cancer and to examine its possible correlation with commonly used prognostic factors, particularly epidermal growth factor receptor (EGFR). Methods: Immunohistochemical analysis was performed on tumors from 194 patients with primary breast cancer. HCRP1 expression was analyzed along with major clinicopathological variables. Results: HCRP1 protein expression was shown to be correlated with age (P = 0.001), histological grade (P = 0.005), tumor progression (P = 0.013), and death (P = 0.001), but not with tumor size, lymph-node metastasis, or Ki67 status. Kaplan-Meier survival curves showed that lower HCRP1 expression was significantly correlated with increased short-term survival (P < 0.001), and both univariate and multivariate analyses revealed that HCRP1, tumor size, lymph-node metastasis, and human epidermal growth factor receptor-2 (HER-2) were independent prognostic factors (all P < 0.05). In addition, low HCRP1 expression was much more frequent in triple negative breast cancer (TNBC; 63.89%) than in luminal (16.95%) and HER-2 overexpression phenotypes (7.5%; P < 0.001), and significant correlations between HCRP1 and survival time were observed for the TNBC group (P < 0.004). Furthermore, an inverse relationship between HCRP1 and EGFR expression was found both for the complete set of all cases (P < 0.001), and for each phenotype analyzed individually (P < 0.05). Conclusion: Our results suggest that HCRP1 may play an important role in EGFR regulation and that its decreased expression is an independent predictor of breast cancer, especially in TNBC patients.     

VEGF-B expression in human primary breast cancers is associated with lymph node metastasis but not angiogenesis

Angiogenesis is essential for tumour growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently VEGF-B, a new VEGF family member that binds to the tyrosine kinase receptor flt-1, has been identified. Although the importance of VEGF has been shown in many human tumour types, the contribution of VEGF-B to tumour neovascularization is unknown in any tumour type. This study therefore measured the mRNA level of VEGF-B and its receptor flt-1 by ribonuclease protection assay and the pattern of VEGF-B expression by immunohistochemistry in 13 normal breast samples and 68 invasive breast cancers. Flt-1 expression was significantly higher in tumours than in normal breast (p=0.02) but no significant difference was seen in VEGF-B between normal and neoplastic breast (p=0.3). There was a significant association between VEGF-B and node status (p=0.02) and the number of involved nodes (p=0.01), but not with age (p=0.7), size (p=0.6), oestrogen receptor (ER) (p=0.2), grade (p=0.5) or vascular invasion (p=0.16). No significant relationship was present between VEGF-B and flt-1 (p=0.2) or tumour vascularity (p=0.4). VEGF-B was expressed mostly in the cytoplasm of tumour cells, although occasional stromal components including fibroblasts and endothelial cells were also positive. No difference in VEGF-B expression was observed adjacent to regions of necrosis, in keeping with this VEGF family member not being hypoxically regulated. These findings suggest that VEGF-B may contribute to tumour progression by a non-angiogenic mechanism, possibly by increasing plasminogen activators and hence metastasis, as has been described in vitro. Measurement of VEGF-B together with other angiogenic factors may identify a poor prognostic patient group, which may benefit from anti-VEGF receptor therapy targeted to flt-1 (VEGFR1) as well as kdr (VEGFR2).     

The expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor is correlated to angiogenesis in breast cancer

It has been shown that human thymidine phosphorylase (TP) Is Identical to platelet-derlved endothelial cell growth factor and has angiogenlc actlvhy. In the present study, the expression of TP was examined In 139 mammary carclnomas and 35 benign mammary disorders using biochemical and lmmunohlstochemlcal methods. Moreover, In order to evaluate the significance of TP expression in mammary carcinomas, the relationship between vascular density and various cllnicopathological factors, including age and menopausal status of patients with a mammary carcinoma, were compared wtth the size, nodal status, expression of estrogen receptor (ER), progesterone receptor (PgR), c-erbB-2, p53 and TP of a mammary carcinoma. Thymidine phosphorylase expression Increased in both the nuclei and cytoplasm of mammary carclnoma cells in comparison to mammary benign disorder cells. The number of mlcroves-sels In mammary carcinomas was generally correlated to the number of tumor cells with TP expression in cytoplasm. The number of cells with TP expression in cytoplasm was significantly large In tumors that measured 34 cm In diameter, compared wtth tumors measuring 1–2 and 5–6 cm in diameter. In mammary tumors of 1–4 cm diameter, TP expression and vessel denslty were slgnlficantly high in tumors negative for ER or positive for cerbB2 and In tumors positive for TP or cerbB2, respectively; whereas tumors of 5–6 cm In diameter were not modified by any cllnlcopathological factors. The results lndlcated that TP plays an Important anglogenetic role In mammary carcinomas, especially tumors with a certain progression.     

Maspin expression in invasive breast cancer: association with other prognostic factors

Maspin is a unique serine protease inhibitor with a molecular weight of 42 kDa. It has been shown to inhibit tumour cell motility and invasion in cell culture, and tumour growth and metastasis in animal models. There is very limited data on the prognostic utility of maspin in human breast cancer. We performed a preliminary study to assess the associations of maspin with other established prognostic factors in invasive breast cancer (IBC). 1068 paraffin-embedded IBCs were immunohistochemically stained with a monoclonal antibody to maspin. A nuclear signal was present in 96% and a cytoplasmic signal in 35% of the cases. Nuclear staining was related to oestrogen (ER) and progesterone receptor (PR) positivity (p < 0.0001), but not to S-phase fraction (SPF) or ploidy. Cytoplasmic staining was related to ER and PR negativity (p < 0.0001), high SPF (p < 0.0001), and aneuploidy (p = 0.003). Thus, maspin nuclear staining was significantly associated with good prognostic factors, while cytoplasmic staining was associated with poor prognostic markers. These findings suggest that the presence of maspin in two different compartments of the cell may have different biological and clinical implications. Additional studies are needed to evaluate further this expression profile of maspin in breast cancer.     

Amplified in breast cancer 1 expression in breast cancer

Aims: The amplified in breast cancer 1 (AIB1), steroid receptor co‐activator family member, acts as an oestrogen receptor (ER) co‐activator. Acting with HER‐2, it is thought to play a role in endocrine resistance by facilitating ER–growth factor crosstalk. The aim was to analyse AIB1 expression by immunohistochemistry and study its correlations with other prognostic variables in breast cancer and its effect on survival. Methods: A tissue microarray comprising tumours from 438 patients with 15.4 years’ median follow‐up was used. Interpretable AIB1 expression obtained in 395 patients was analysed along with other prognostic factors in breast cancer. Results: AIB1 expression scores ranged from 0 to 30; positive AIB1 expression (score > 14) was seen in 146/395 breast cancers; it correlated negatively with ER (P = 0.003) and progesterone receptor (PR) (P = 0.007), and positively with HER‐2 (P = 0.005) and tumour grade (P = 0.014). It did not correlate with nodal status (P = 0.437). Among ER+ patients, AIB1 expression showed a trend towards loss of PR expression (29% versus 20%; P = 0.14). AIB1 did not predict survival on univariate or multivariate analysis. Conclusions: AIB1 expression correlates with HER‐2 expression in breast cancer and shows a trend of association with loss of PR expression in ER+ tumours. Our study supports the postulated role of AIB1 in ER–growth factor interactions.     

Preserved Axin expression is associated with an aggressive phenotype in invasive breast carcinomas

Reduced Axin expression has been associated with an aggressive behavior in lung and esophageal squamous cell carcinomas. Its role in breast cancer has not been defined. The aim of our study was to investigate the expression pattern of Axin protein in invasive breast carcinomas, in relation to the behavior and prognosis of the disease. Immunohistochemistry was performed for the detection of Axin expression in 232 breast cancer tissues. Univariate and multivariate statistical analyses were used to assess the relation of Axin expression with classic clinicopathological parameters, patients' survival and various biologic markers Human Epidermal Factor‐2 (HER‐2), Ki‐67, topoIIα, glycogen synthase kinase‐3β (GSK‐3β)]. Preserved cytoplasmic Axin expression was positively correlated to lymph node invasion, HER‐2 and GSK‐3β and inversely to Ki‐67 and topoIIα. Nuclear Axin was positively associated with tumor size. Stromal Axin showed a parallel association with lymph node status and HER‐2. In the subgroup of lobular breast carcinomas, preserved Axin was found to exert an unfavorable impact on patients' overall survival. Our findings indicate, for the first time, that in invasive breast cancer preserved Axin expression is associated with a more aggressive phenotype and that in the discrete subtype of lobular breast carcinomas Axin negatively influences patients' overall survival.     

Expression of c-ets-1 mRNA is associated with an invasive, EMT-derived phenotype in breast carcinoma cell lines

We have previously observed in vitro that some stromal proteinases (MMP-2, MT1-MMP) were expressed or activated by invasive carcinoma cell lines exhibiting mesenchymal features, presumably acquired through an epithelial to mesenchymal transition (EMT). To examine the potential contribution of c-ets-1 to this phenotype, we have compared here the expression of c-ets-1 with invasiveness in vitro and expression of vimentin, E-cadherin, uPA, MMP-1 and MMP-3 in a panel of human breast cancer cell lines. Our results clearly demonstrate an association between c-ets-1 expression and the invasive, EMT-derived phenotype, which is typified by the expression of vimentin and the lack of E-cadherin. While absent from the two non-invasive, vimentin-negative cell lines, c-ets-1 was abundantly expressed in all the four vimentin-positive lines. However, we could not find a clear quantitative or qualitative relationship between the expression of c-ets-1 and the three proteinases known to be regulated by c-ets-1, except that when they were expressed, it was only in the invasive c-ets-1-positive lines. UPA mRNAs were found in three of the four vimentin-positive lines, MMP-1 in two of the four, and MMP-3 could not be detected in any of the cell lines. Intriguingly, MDA-MB-435 cells, which exhibit the highest metastatic potential of these cell lines in nude mice, expressed vimentin and c-ets-1, but lacked expression of these three proteinases, at least under the culture conditions employed. Taken together, our results show that c-ets-1 expression is associated with an invasive, EMT-derived phenotype in breast cancer cells, although it is apparently not sufficient to ensure the expression of uPA, MMP-1 or MMP-3, in the vimentin-positive cells. Such proteases regulation is undoubtedly qualified by the cellular context. This study therefore advances our understanding of the molecular regulation of invasiveness in EMT-associated carcinoma progression, and suggests that c-ets-1 may contribute to the invasive phenotype in carcinoma cells.     

High Notch1 protein expression is an early event in breast cancer development and is associated with the HER‐2 molecular subtype

Zardawi S J, Zardawi I, McNeil C M, Millar E K A, McLeod D, Morey A L, Crea P, Murphy N C, Pinese M, Lopez‐Knowles E, Oakes S R, Ormandy C J, Qiu M R, Hamilton A, Spillane A, Soon Lee C, Sutherland R L, Musgrove E A & O’Toole S A
(2010) Histopathology 56, 286–296 High Notch1 protein expression is an early event in breast cancer development and is associated with the HER‐2 molecular subtype Aims: Activation of Notch signalling results in hyperplasia and tumorigenesis in murine mammary epithelium. However, there is little information regarding the expression of Notch1 in premalignant lesions and early breast cancer. We investigated expression of Notch1 in breast cancer development and its association with molecular subtypes. Methods and results: Immunohistochemical expression of Notch1 was determined in a murine model of mammary carcinogenesis and in breast tissue from two cohorts of breast cancer patients, the first (n = 222) comprising a histological progression series and the second an outcome series of 228 patients with operable invasive ductal carcinoma. Enhanced expression of Notch1 protein was an early event in both murine and human breast cancer development with progressive increases in expression with the development of hyperplasia and malignancy. High Notch1 was not prognostic in the outcome cohort. There was, however, a highly significant association of high Notch1 protein with the HER‐2 molecular subtype of breast cancer (P = 0.008). Conclusions: These data demonstrate that aberrant Notch regulation is an early event in mammary carcinogenesis and is associated with the HER‐2 molecular subtype of breast cancer, and suggest the Notch signalling pathway may be a potential therapeutic target worthy of further investigation.     

Fibroblast growth factor 8 induced downregulation of thrombospondin 1 is mediated by the MEK/ERK and PI3K pathways in breast cancer cells

Expression of fibroblast growth factor 8 (FGF-8) is increased in several forms of hormonal cancer. It was previously shown to regulate expression of thrombospondin 1 (TSP-1), an inhibitor of angiogenesis, in S115 breast cancer cells. Here, we studied the FGF-8-activated signalling pathways mediating TSP-1 repression in S115 cells and in non-tumorigenic MCF10A cells. Inhibition of FGF receptors or of MEK1/2 and PI3K with specific inhibitors (PD173074, U0126 or LY294002, respectively) restored TSP-1 mRNA expression in the presence of FGF-8 in S115 cells. Furthermore, U0126 and LY294002 increased TSP-1 mRNA expression in S115 cells over-expressing FGF-8. In MCF10A cells, FGF-8 treatment also decreased TSP-1 expression and the effect was dependent on active MEK1/2. In conclusion, FGF-8 suppresses TSP-1 expression through two independent pathways, MEK1/2 and PI3K. Repression of TSP-1 may be an important mechanism involved in induction of an angiogenic phenotype and growth of FGF-8-expressing breast cancer.