P van der Groep

Twist contributes to hormone resistance in breast cancer by downregulating estrogen receptor-α

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.

Distinction between hereditary and sporadic breast cancer on the basis of clinicopathological data.

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.

Hypoxia-inducible factor-1α expression requires PI 3-kinase activity and correlates with Akt1 phosphorylation in invasive breast carcinomas

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.

Progressive derailment of cell cycle regulators in endometrial carcinogenesis

Background: Derailments of the control mechanisms of the cell 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.

Fibroblast growth factor receptors in breast cancer: expression, downstream effects, and possible drug targets

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.

Molecular profile of ductal carcinoma in situ of the breast in BRCA1 and BRCA2 germline mutation carriers

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.

Discordance in ERα, PR and HER2 receptor status across different distant breast cancer metastases within the same patient.

BACKGROUND We studied discordance in estrogen receptor alpha (ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status between multiple distant metastases from the same breast cancer patient. MATERIAL AND METHODS Multiple distant metastases from 55 female patients were stained for ERα, PR and HER2 by immunohistochemistry and in situ hybridization for confirmation of the HER2 status. RESULTS Different metastatic sites within the same patient showed discordance in ERα receptor status in 7.3% or 10.9% of patients (using a 10% or 1% threshold for positivity, respectively). For PR, 29.1% or 30.9% of patients showed discordance. Taking ERα and PR together, 36.4% of cases (both thresholds) showed discrepancy between metastases. In 10.9% (10% threshold) or 14.5% of patients (1% threshold), such discordance could have clinical consequences with regard to hormonal treatment. For HER2, there was 3.6% discordance on the immunohistochemical level but 0% on the gene level. CONCLUSION In a significant proportion of metastatic breast cancer patients, discordance in ERα and PR receptor status between different metastatic sites was observed. This implies that multiple metastases may need to be biopsied to optimally reassess receptors.

Hypoxia‐inducible factor 1α is essential for hypoxic p27 induction in endometrioid endometrial carcinoma

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

Abstract P2-06-02: Genomic evolution from primary breast cancers to distant metastases

Purpose: Genomic evolution is an accepted concept during cancer development and progression. However, little is known about the changes that occur during the metastatic process despite the fact that breast cancer metastases are the leading cause of death in breast cancer patients. The purpose of this study was therefore to investigate whether distant breast cancer metastases show progression in copy number changes of onco- and tumor suppressor genes compared to their primary tumor. Material and methods: Multiplex ligation-dependent probe amplification (MLPA) was used to compare copy number of 21 established oncogenes and tumor suppressor genes between primary breast cancer samples and corresponding distant metastases in 55 patients. Genes studied were ESR1, EGFR, FGFR1, ADAM 9, IKBKB, PRDM14, MTDH, MYC, CCND1, EMSY, CDH1, TRAF4, CPD, MED1, HER2, CDC6, TOP2A, MAPT, BIRC5, CCNE1 and AURKA . Results: Overall, there was no significant difference in mean copy number between primary tumors (1.26 +/− 0.6) and metastases (1.27 +/− 0.64) (p = 0.826), but on the individual gene level, mean copy number for PRDM14 ( p = 0,023), MED1 ( p = 0.001) and CCNE1 ( p = 0.039) were significantly higher while TRAF4 ( p = 0.038) copy number was significantly lower in the metastases. Using dichotomized MLPA data, MTDH amplifications were significantly more frequent in the primary cancers compared to the metastases (27.3% vs.14.6%, p = 0.039), while significantly more CDH1 losses were found in the metastases compared to the primary tumor (23.6% vs. 9.1%, p = 0.039). Conclusion: Distant breast cancer metastases show genomic evolution from the primary cancer as reflected by copy number changes in several established onco- and tumor suppressor genes. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-06-02.

Lymphovascular Invasion in BRCA Related Breast Cancer Compared to Sporadic Controls

Background Germline mutations in the BRCA1 gene predispose to the development of breast cancer, exhibiting a specific histological phenotype. Identification of possible hallmarks of these tumors is important for selecting patients for genetic screening and provides inside in carcinogenetic pathways. Since BRCA1-associated breast cancers have pushing borders that prevent them from easily reaching vessels and are often of the medullary (like) type that is known to have a low rate of lympho-vascular invasion (LVI), we hypothesized that absence of LVI could characterize BRCA1 related breast cancer.