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Cancer treatments are increasingly focusing on the molecular mechanisms underlying the oncogenic processes present in tumors of individual patients. Fibroblast growth factor receptors (FGFRs) are among the many molecules that are involved in oncogenesis and are currently under investigation for their potential as drug targets in breast cancer patients. These receptor tyrosine kinases play a role in several processes including proliferation, angiogenesis, and migration. Alterations in these basal processes can contribute to the development and progression of tumors. Among breast cancer patients, several subgroups have been shown to harbor genetic aberrations in FGFRs, including amplifications of FGFR1, FGFR2, and FGFR4 and mutations in FGFR2 and FGFR4. Here, we review in vitro and in vivo models that have partly elucidated the molecular implications of these different genetic aberrations, the resulting tumor characteristics, and the potential of FGFRs as therapeutic targets for breast cancer treatment. [ABSTRACT FROM AUTHOR]

The role of estrogen receptor-α (ER) in breast cancer development, and as a primary clinical marker for breast cancer prognosis, has been well documented. In this study, we identified the oncogenic protein, TWIST1 (Twist), which is overexpressed in high-grade breast cancers, as a potential negative regulator of ER expression. Functional characterization of ER regulation by Twist was performed using Twist low (MCF-7, T-47D) and Twist high (Hs 578T, MDA-MB-231, MCF-7/Twist) expressing cell lines. All Twist high expressing cell lines exhibited low ER transcript and protein levels. By chromatin immunoprecipitation and promoter assays, we demonstrated that Twist could directly bind to E-boxes in the ER promoter and significantly downregulate ER promoter activity in vitro. Functionally, Twist overexpression caused estrogen-independent proliferation of breast cells, and promoted hormone resistance to the selective estrogen receptor modulator tamoxifen and selective estrogen receptor down-regulator fulvestrant. Importantly, this effect was reversible on downregulating Twist. In addition, orthotopic tumors generated in mice using MCF-7/Twist cells were resistant to tamoxifen. These tumors had high vascular volume and permeability surface area, as determined by magnetic resonance imaging (MRI). Mechanistically, Twist recruited DNA methyltransferase 3B (DNMT3B) to the ER promoter, leading to a significantly higher degree of ER promoter methylation compared with parental cells. Furthermore, we demonstrated by co-immunoprecipitation that Twist interacted with histone deacetylase 1 (HDAC1) at the ER promoter, causing histone deacetylation and chromatin condensation, further reducing ER transcript levels. Functional re-expression of ER was achieved using the demethylating agent, 5-azacytidine, and the HDAC inhibitor, valproic acid. Finally, an inverse relationship was observed between Twist and ER expression in human breast tumors. In summary, the regulation of ER by Twist could be an underlying mechanism for the loss of ER activity observed in breast tumors, and may contribute to the generation of hormone-resistant, ER-negative breast cancer. [ABSTRACT FROM AUTHOR]

AIMS: Ductal carcinoma in situ (DCIS) is an established late precursor of sporadic invasive breast cancer and to a large extent parallels its invasive counterpart with respect to molecular changes and immunophenotype. Invasive breast cancers in germline BRCA1 and BRCA2 mutation carriers have a distinct "basal" and "luminal" immunophenotype, respectively, but the immunophenotype of their precursor lesions has hardly been studied, and this was the aim of this study. METHODS: DCIS lesions of 25 proven BRCA1 and 9 proven BRCA2 germline mutation carriers and their 22 and 6, respectively, accompanying invasive lesions were stained by immunohistochemistry for oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER)2/neu, cytokeratin (CK)5/6, CK14, epidermal growth factor receptor (EGFR) and Ki67. RESULTS: DCIS lesions in BRCA1 mutation carriers were mostly of the basal molecular type with low ER/PR/HER2 expression, while they frequently expressed CK5/6, CK14 and EGFR, and were mostly grade 3 and highly proliferative. DCIS lesions in BRCA2 mutation carriers were mostly of luminal molecular type with frequent expression of ER/PR, and infrequent expression of CK5/6, CK14 and EGFR, and they were mostly grade 3 and showed low proliferation. In BRCA1 and BRCA2 mutation carriers there was a high concordance between DCIS lesions and their concomitant invasive counterpart with regard to expression of individual markers as well as "molecular" subtype. CONCLUSIONS: Although the number of cases studied was low, DCIS lesions in BRCA1 and BRCA2 mutations carriers are usually of the basal and luminal molecular type, respectively, similar to their accompanying invasive cancers, thereby providing evidence that DCIS is a direct precursor lesion in these hereditary predisposed patients. This also suggests that crucial carcinogenetic events leading to these phenotypes in hereditary predisposed patients occur before the stage of invasion. [ABSTRACT FROM AUTHOR]

Background: Derailments of the control mechanisms of the celll cycle can initiate carcinogenesis, and play a role in progression to cancer. Aim: To explore the expression of cell cycle proteins in normal, premalignant and malignant endometrial lesions representing the morphologically well defined stepwise model of human endometrial carcinogenesis Methods: Observational study. Paraffin-embedded specimens from inactive endometrium (n = 16), endometrial hyperplasia (n = 23) and endometrioid endometrial carcinoma (n = 39) were stained immunohistochemically for cyclin A, cyclin B1, cyclin D1, cyclin E, cdk2, p16, p21, p27, p53 and Ki67(MIB-1)). Differences in expression between the tissues, and correlation with classical prognostic factors for the carcinomas were analysed. Results: Expression of cyclin A and Ki67 gradually increased from normal through hyperplasia to carcinoma, indicating that proliferation increases over the carcinogenetic spectrum, cdk2, p16 and p21 gradually increased from normal through hyperplasia to carcinoma, indicating their potential importance in both early and late carcinogenesis. Cyclin D1, cyclin E and p53 especially increased and p27 decreased from hyperplasia to carcinoma, underlining their role in late carcinogenesis. In cancers, expression of cyclin A, p53 and Ki67 was positively correlated to grade, and cyclin A was positively correlated with cdk2, p21, Ki67, cyclin E and p53. Conclusion: During (endometrioid) endometrial carcinogenesis, there is increasing proliferation paralleled by progressive derailment of cyclin B1, cyclin D1, cyclin E, p16, p21, p27, p53, and cdk2, indicating the importance of these cell cycle regulators in endometrial carcinogenesis. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor 1α (HIF-1α) plays an essential role in the adaptive response of cells to hypoxia. The cyclin-dependent kinase inhibitor p27(Kip1) is highly expressed in the normal endometrium but is lost during endometrial carcinogenesis. However, in high-grade cancers, p27 re-expression is observed. We analysed the role of HIF-1α in hypoxia-induced expression of p27 in vitro and in vivo in endometrial cancer. Paraffin-embedded specimens from endometrioid endometrial carcinoma ( n = 39) were stained immunohistochemically for HIF-1α, p27, and Ki67. HEC1B, an endometrial carcinoma cell line, was cultured under normoxic or hypoxic conditions in the presence or absence of transiently expressed short hairpin RNAs targeting HIF-1α. Protein expression of p27 and HIF-1α was assessed by western blotting. Immunohistochemical staining revealed perinecrotic HIF-1α expression in 67% of the cases and p27 staining centrally in the tumour islands, mostly around necrosis, in 46% of the cases. In 50% of the tumours with perinecrotic HIF-1α expression, p27 and HIF-1α perinecrotic/central co-localization was observed. In these tumour sections, hypoxia-associated p27 expression showed less proliferation around necrosis. Analysis of cultured endometrial carcinoma cells demonstrated that p27 protein expression is induced by hypoxia. This induction was abrogated by transient knockdown of HIF-1α using RNAi. Furthermore, hypoxia induced cell cycle arrest in HEC1B cells. We conclude that, in endometrioid endometrial carcinoma, p27 re-expression by hypoxia is HIF-1α-dependent and leads to cell cycle arrest. This may contribute to the survival of cancer cells in hypoxic parts of the tumour. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor-1 alpha (HIF-1α) is the regulatory subunit of the heterodimeric transcription factor HIF-1 and the key factor in cellular response to low oxygen tension. Expression of HIF-1α protein is associated with poor patient survival and therapy resistance in many types of solid tumors. Insight into HIF-1α regulation in solid tumors is important for therapeutic strategies. In this study, we determined the pathophysiological relevance of HIF-1α regulation by the oncogenic phosphatidylinositol 3′-kinase (PI 3-kinase)/Akt signaling pathway. We modeled the physiology of hypoxic tumor regions by culturing carcinoma cells under low oxygen tension in the absence of serum. We observed that hypoxic induction of HIF-1α protein was decreased by serum deprivation. Overexpression of dominant-active Akt1 restored HIF-1α expression, whereas inhibition of PI 3-kinase activity reduced hypoxic HIF-1α protein levels to a similar extent as serum deprivation. Immunohistochemical analysis of 95 human breast cancers revealed that lack of Akt1 phosphorylation correlates with low HIF-1α levels. To our knowledge, this is the first reported comparison between HIF-1α expression and Akt phosphorylation in human carcinomas. We conclude that Akt activity is physiologically relevant for HIF-1α expression in breast cancer. This implies that HIF-1α function might be therapeutically targeted by inhibition of the PI 3-kinase/Akt pathway.Oncogene (2006) 25, 6123–6127. doi:10.1038/sj.onc.1209643; published online 8 May 2006 [ABSTRACT FROM AUTHOR]

Background: About 5% of all breast cancer cases are attributable to germline mutations in BRCA1 or BRCA2 genes. BRCA mutations in suspected carriers, however, may be missed, which hampers genetic counselling. Materials and methods: Different clinicopathological features were compared between 22 breast cancers from carriers of proved BRCA1 mutations and 604 cancers from sporadic controls. In addition, 5 BRCA2- related breast cancers and 66 breast cancers of untested patients at intermediate risk and 19 breast cancers of untested patients at high risk of hereditary disease on the basis of family history were evaluated. Results: A ‘probably sporadic’ class (age ⩾54 years and epidermal growth factor receptor (EGFR) negative; 68% of cases) with a 0% chance of BRCA1-related breast cancer containing 79% of the sporadic cases was yielded by using a decision tree with age, Ki67 and EGFR. A 75% chance of BRCA1-related breast cancer was shown by the ‘probably BRCA1-related’ class (age <54 years and Ki67 ⩾25%; 8% of cases) with 82% of the BRCA1-related cases but only 1 .4% of the sporadic cases. Most cases at intermediate or high risk of hereditary disease on the basis of family history could be classified with high probability as either probably BRCA1 related or probably sporadic. Conclusion: Breast carcinomas can be classified with a high level of certainty as sporadic or related to BRCA1 germline mutations by using a decision tree with age, Ki67 and EGFR. This can be clinically useful in mutation analysis in families with a borderline risk of hereditary disease. [ABSTRACT FROM AUTHOR]

Activation of the Wnt signaling pathway initiates the transformation of colorectal epithelial cells, although the transition to metastatic cancer requires angiogenesis. We have investigated the expression of the von Hippel–Lindau (VHL) tumor suppressor in the intestines from humans and mice. Here, we show that VHL expression is regulated by TCF4 and is restricted to the proliferative compartment at the bottom of intestinal crypts. Accordingly, VHL is completely absent from the proliferative intestinal pockets of Tcf4−/− perinatal mice. We observed complementary staining of the hypoxia-inducible factor (HIF) 1α to VHL in normal intestinal epithelium as well as in all stages of colorectal cancer (CRC). To the best of our knowledge, this is the first report demonstrating the presence of nuclear HIF1α in normoxic healthy adult tissue. Although we observed upregulated levels of VHL in very early CRC lesions from sporadic and familial adenomatous polyposis patients – presumably due to activated Wnt signaling – a clear reduction of VHL expression is observed in later stages of CRC progression, coinciding with stabilization of HIF1α. As loss of VHL in later stages of CRC progression results in stabilization of HIF, these data provide evidence that selection for VHL downregulation provides a proangiogenic impulse for CRC progression.Oncogene (2006) 25, 3065–3070. doi:10.1038/sj.onc.1209330; published online 9 January 2006 [ABSTRACT FROM AUTHOR]

Background: Intratumorous hypoxia triggers a broad cellular response mediated by the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1α concentrations increase during breast carcinogenesis, and are associated with poor prognosis. An earlier study noted two HIF-1α overexpression patterns: diffuse scattered throughout the tissue and confined to perinecrotic cells. Aims: To investigate the prognostic impact of these different HIF-1α overexpression patterns in relation to its downstream effectors carbonic anhydrase (CA) IX and glucose transporter 1 (GLUT-i). Methods: HIF-1α, CA IX, and GLUT-1 expression was studied by immunohistochemistry, including double staining for CA IX and HIF-1α. Clinical data included disease free survival, lymph node status, and tumour size. Results: HIF-1α overexpression (44% of cases) had a perinecrotic (13.5%) or diffuse staining pattern (30.5%). CA IX expression was detectable in 12.5% of breast cancers, whereas GLUT-1 expression was seen in 29%, with both showing perinecrotic membrane staining. Perinecrotic HIF-1α overexpression was highly associated with CA IX and GLUT-1 overexpression, and double staining for HIF-1α and CA IX re showed strong expression in the same cells. Diffusely overexpressed HIF-1α was not associated with CA IX or GLUT-1 expression. Patients with diffuse HIF-1α staining had a significantly better prognosis than patients with perinecrotically overexpressed HIF-1α. Conclusions: Different regulation pathways of HIF-1α overexpression exist in breast cancer: (1) hypoxia induced, perinecrotic HIF-1α overexpression with strong expression of hypoxia associated genes (CA IX and GLUT-1), which is associated with a poor prognosis; and (2) diffuse HIF-1α overexpression lacking major hypoxia associated downstream effects, resulting in a more favourable prognosis. [ABSTRACT FROM AUTHOR]

Bos R, van Diest P J, de Jong J S, van der Groep P, van der Valk P&van der Wall E(2005)Histopathology46,31–36Hypoxia-inducible factor-1α is associated with angiogenesis, and expression of bFGF, PDGF-BB, and EGFR in invasive breast cancer: Hypoxia-inducible factor-1 (HIF-1) is the key transcription factor regulating the cellular response to hypoxia, including angiogenesis. Growth factors play an important role in tumour growth and angiogenesis and some have been shown to be induced by HIF-1in vitro. This study investigated if angiogenesis or growth factors or their receptors are associated with HIF-1α in invasive breast cancer.: High levels of HIF-1α, detected by immunohistochemistry in 45 breast cancers, were positively associated with increased microvessel density (as a measure of angiogenesis) (P = 0.023). Furthermore, high levels of HIF-1α were associated with epithelial expression (≥ 10%) of epidermal growth factor receptor (EGFR) (P = 0.011), platelet-derived growth factor (PDGF)-BB (P < 0.001), and basic fibroblast growth factor (bFGF) (P = 0.045). A positive, yet insignificant, trend for HIF-1α to be associated with epithelial expression of transforming growth factor (TGF)-α (P = 0.081) and vascular endothelial growth factor (VEGF) (P = 0.109) was noticed as well as an inverse association with stromal expression of TGF-β-R1 (P = 0.070).: In invasive breast cancer, HIF-1α is associated with angiogenesis, and expression of growth factors bFGF and PDGF-BB, and the receptor EGFR. Thus, agents targeting HIF-1 may combine different pathways of inhibiting breast cancer growth, including angiogenesis and growth factors. [ABSTRACT FROM AUTHOR]

Background: Recent evidence suggests that functional intratumorous lymph vessels may be absent from some human cancers. This could result from either the failure of tumours to induce lymphangiogenesis, or the collapse of lymph vessels, caused by high interstitial tumour pressure. Methods: To differentiate between these two hypotheses, paraffin wax embedded clinical specimens from normal breast (n = 13), usual ductal hyperplasia (n = 11), ductal carcinoma in situ (n = 21), and invasive breast cancer (n = 40) were compared for lymphatic and blood vessel density by immunohistochemistry with antibodies to the lymphatic endothelial hyaluronan receptor (LYVE-1) and CD3 1, respectively. Results: Lymph vessel density was tower than blood vessel density in normal breast tissue. Within breast lobuli, lymph vessels were absent. In premalignant lesions blood microvesset density increased, whereas no increase in lymph vessels could be seen intralesionally. In invasive cancers, lymph vessels were absent in all but a few cases, where probably some pre-existing lymph vessels remained, although blood microvessel density was once again increased. Conclusion: Unlike angiogenesis, lymphangiogenesis is absent during breast carcinogenesis. This, and not rising interstitial pressure caused by an increase in the size of lesions, explains the absence of infratumorous lymph vessels in invasive breast cancer. [ABSTRACT FROM AUTHOR]

Background: Hypoxia inducible factor 1alpha (HIF-1alpha) plays an essential role in the adaptive response of cells to hypoxia and is associated with aggressive tumour behaviour. We have shown p27kip1, which is generally reduced in endometrial cancer, to be re-expressed in hypoxic regions. This possibly contributes to survival of cancer cells. The aim of this study was to evaluate the prognostic value of HIF-1alpha and p27kip expression in patients with endometrioid endometrial cancer.Methods: Expression levels of HIF-1alpha, CAIX, Glut-1, and p27kip1 were analyzed by immunohistochemistry. Percentage of positive cells, staining pattern (perinecrotic, diffuse, or mixed) and presence of necrosis were noted.Results: Necrosis was correlated with shortened disease free survival (DFS) (p = 0.008) and overall survival (OS) (p = 0.045). For DFS, perinecrotic HIF-1alpha expression was also prognostic (p = 0.044). Moreover, high p27kip1 expression was an additional prognostic factor for these patients with perinecrotic HIF-1alpha expression. In multivariate Cox regression, perinecrotic HIF-expression emerged as an independent prognostic factor. Perinecrotic HIF-1alpha expression was significantly associated with CAIX and Glut-1 expression, pointing towards functional HIF-1.Conclusions: In patients with endometrioid endometrial cancer, necrosis and necrosis-related expression of HIF-1alpha are important prognostic factors. More aggressive adjuvant treatment might be necessary to improve the outcome of patients with these characteristics. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about breast cancer.