E. M. J. J. Berns

miR-141 regulates KEAP1 and modulates cisplatin sensitivity in ovarian cancer cells

Epithelial ovarian cancer is the most lethal gynecological malignancy in the Western world. A major impediment for the successful treatment is the development of drug resistance. The molecular processes that contribute to resistance have been extensively studied; however, there is not much known about regulation by microRNAs (miRNAs). We compared miRNA expression profiles of an isogenic cisplatin-sensitive and -resistant ovarian cancer cell line pair (A2780/A2780 DDP) and found 27 miRNAs to be differentially expressed (⩾2-fold). Five of these, including the family members miR-141/200c, showed a correlation with cisplatin sensitivity in the NCI-60 panel. Overexpression of miR-141 resulted in enhanced resistance to cisplatin in ovarian cancer cell lines. We next correlated the expression level of miR-141 in 132 primary ovarian tumors (108 serous and 24 non-serous) with response to platinum-based chemotherapy. Although no differences were observed in the serous tumors, miR-141 levels were higher in non-serous ovarian tumors that did not respond well to therapy (platinum-free interval <6 months). We demonstrate that miR-141 directly targets KEAP1, and that downregulation of KEAP1 induces cisplatin resistance. Conversely, overexpression of KEAP1 significantly enhanced cisplatin sensitivity. Expression of KEAP1 with its 3′-UTR, and a 3′-UTR in which the miR-141 target site has been mutated, revealed that miR-141 regulates KEAP1 upon exposure to cisplatin. Finally, we show that the NF-κB pathway, which can be regulated by KEAP1, is activated upon miR-141 overexpression, and that inhibition of this pathway partially reverses miR-141-mediated cisplatin resistance. These findings demonstrate that the miR-141-mediated regulation of KEAP1 has a crucial role in the cellular response to cisplatin.

Mutations in residues of TP53 that directly contact DNA predict poor outcome in human primary breast cancer

The tumour-suppressor gene TP53 is frequently mutated in breast tumours, and the majority of the mutations are clustered within the core domain, the region involved in DNA binding. We searched for alterations in this central domain of the TP53gene in 222 human breast cancer specimens using polymerase chain reaction-single-strand conformation analysis (PCR-SSCA) followed by sequencing. TP53 gene mutations were observed in 66 tumours (31%), including three tumours that contain two mutations. Fifty-four (78%) of these mutations were missense point mutations, one was a nonsense mutation and four were deletions and/or insertions causing disruption of the protein reading frame, whereas four mutations were either silent or a polymorphism (at codon 213; n = 6). Interestingly, the majority of missense mutations were observed at codon 248. The outcome has been related with patient and tumour characteristics, and with prognosis in 177 patients who were eligible for analysis of both relapse-free and overall survival (median survival for patients alive was 115 months). There was no significant association between the frequency of TP53 mutations and menopausal or nodal status, or tumour size. In a Cox univariate analysis, TP53 gene mutation was significantly associated with poor relapse-free survival (RFS: P = 0.02) but not with overall survival (OS: P = 0.07). In a Cox multivariate analysis, including classical prognostic factors, TP53 gene mutation independently predicted poor RFS and OS (RHR = 1.8 and 1.6 respectively). Unexpectedly, the median relapse-free survival of patients with a polymorphism at codon 213 or with a silent mutation was shorter (median 11 months) than the median relapse-free survival of patients with or without a TP53 gene mutation (median 34 or 48 months respectively). In an exploratory subset analysis, mutations in codons that directly contact DNA were related with the poorest relapse-free (P < 0.05) and overall survival (P < 0.02). These data imply that in the analysis of the prognostic value of TP53, the type of mutation and its biological function should be considered.

The VEGF pathway and the AKT/mTOR/p70S6K1 signalling pathway in human epithelial ovarian cancer

Vascular endothelial growth factor (VEGF)-A inhibitors exhibit unseen high responses and toxicity in recurrent epithelial ovarian cancer suggesting an important role for the VEGF/VEGFR pathway. We studied the correlation of VEGF signalling and AKT/mTOR signalling. Using a tissue microarray of clinical samples (N=86), tumour cell immunohistochemical staining of AKT/mTOR downstream targets, pS6 and p4E-BP1, together with tumour cell staining of VEGF-A and pVEGFR2 were semi-quantified. A correlation was found between the marker for VEGFR2 activation (pVEGFR2) and a downstream target of AKT/mTOR signalling (pS6) (R=0.29; P=0.002). Additional gene expression analysis in an independent cDNA microarray dataset (N=24) showed a negative correlation (R=−0.73, P<0.0001) between the RPS6 and the VEGFR2 gene, which is consistent as the gene expression and phosphorylation of S6 is inversely regulated. An activated tumour cell VEGFR2/AKT/mTOR pathway was associated with increased incidence of ascites (χ2, P=0.002) and reduced overall survival of cisplatin–taxane-based patients with serous histology (N=32, log-rank test, P=0.04). These data propose that VEGF-A signalling acts on tumour cells as a stimulator of the AKT/mTOR pathway. Although VEGF-A inhibitors are classified as anti-angiogenic drugs, these data suggest that the working mechanism has an important additional modality of targeting the tumour cells directly.

Reduced expression of BAX is associated with poor prognosis in patients with epithelial ovarian cancer: A multifactorial analysis of TP53, p21, BAX and BCL-2

Traditional clinicopathological features do not predict which patients will develop chemotherapy resistance. The TP53 gene is frequently altered in ovarian cancer but its prognostic implications are controversial. Little is known on the impact of TP53-downstream genes on prognosis. Using molecular and immunohistochemical analyses we examined TP53 and its downstream genes p21 BAX and BCL-2 in ovarian tumour tissues and have evaluated the results in relation to clinico-pathological parameters, clinical outcome and response to platinum-based chemotherapy. Associations of tested factors and patient and tumour characteristics were studied by Spearman rank correlation and Pearsons χ2 test. The Cox proportional hazard model was used for univariate and multivariate analysis. The associations of tested factors with response was tested using logistic regression analysis. TP53 mutation, p21 and BCL-2 expression were not associated with increased rates of progression and death. Expression of TP53 was associated with a shorter overall survival only (relative hazard rate [RHR] 2.01 P= 0.03). Interestingly, when combining TP53 mutation and expression data, this resulted in an increased association with overall survival (P = 0.008). BAX expression was found to be associated with both progression-free (RHR 0.44 P = 0.05) and overall survival (RHR 0.42 P = 0.03). Those patients who simultaneously expressed BAX and BCL-2 had a longer progression-free and overall survival compared to patients whose tumours did not express BCL-2 (P = 0.05 and 0.015 respectively). No relations were observed between tested factors and response to platinum-based chemotherapy. We conclude that BAX expression may represent a prognostic indicator for patients with ovarian cancer and that the combined evaluation of BAX and BCL-2 may provide additional prognostic significance.

Large regional differences in the frequency of distinct BRCA1/BRCA2 mutations in 517 Dutch breast and/or ovarian cancer families

In 517 Dutch families at a family cancer clinic, we screened for BRCA1/2 alterations using the Protein Truncation Test (PTT) covering approximately 60% of the coding sequences of both genes and direct testing for a number of previously identified Dutch recurrent mutations. In 119 (23%) of the 517 families, we detected a mutation in BRCA1 (n=98; 19%) or BRCA2 (n=21; 4%). BRCA1/2 mutations were found in 72 (52%) of 138 families with breast and ovarian cancer (HBOC), in 43 (13%) of the 339 families with breast cancer only (HBC), in 4 (36%) of 11 families with ovarian cancer only (HOC), and in nine of 29 families with one single young case (<40 years) of breast cancer. Between the different subgroups of families (subdivided by the number of patients, cancer phenotype and age of onset) the proportion of BRCA1/2 mutations detected, varied between 6 and 82%. Eight different mutations, each encountered in at least six distinct families, represented as much as 61% (73/119 families) of all mutations found. The original birthplaces of the ancestors of carriers of these eight recurrent mutations were traced. To estimate the relative contribution of two important regional recurrent mutations (BRCA1 founder mutation IVS12-1643del3835 and BRCA2 founder mutation 5579insA) to the overall occurrence of breast cancer, we performed a population-based study in two specific small regions. The two region-specific BRCA1 and BRCA2 founder mutations were detected in 2.8% (3/106) and 3.2% (3/93) of the unselected breast tumours, respectively. Of tumours diagnosed before the age of 50 years, 6.9% (3/43) and 6.6% (2/30) carried the region-specific founder mutation. Thus, large regional differences exist in the prevalence of certain specific BRCA1/BRCA2 founder mutations, even in very small areas concerning populations of approximately 200000 inhabitants.

p53 protein accumulation predicts poor response to tamoxifen therapy of patients with recurrent breast cancer.

PURPOSE Mutations of the p53 gene are frequently observed in primary breast cancer and accumulation of p53 protein has been used as a surrogate marker of p53 inactivation. Previous studies have shown that p53 accumulation is related to poor prognosis in primary breast cancer. We studied whether p53 protein accumulation is a predictive factor for response to tamoxifen treatment in patients with recurrent breast cancer. PATIENTS AND METHODS Levels of p53, estrogen receptor (ER), progesterone receptor (PgR), and urokinase-type plasminogen activator (uPA) were assayed in cytosolic extracts derived from primary tumors of 401 tamoxifen-naive patients who developed recurrent disease. All patients in the study received tamoxifen therapy upon relapse (median follow-up, 69 months). Association of tested factors with response to tamoxifen treatment was studied by logistic regression analysis, and with survival after the start of treatment by Cox univariate and multivariate regression analysis. RESULTS p53 levels (median, 0.23 ng/mg protein) were not related to ER or PgR levels, but positively correlated with uPA (P < .0001). In a test for trend, we observed an association of p53 protein levels with response to tamoxifen therapy. When dichotomized (at the median value), 42% in the p53-high versus 56% in the p53-low group showed a response. In multivariate analysis, including patient and tumor characteristics, p53 accumulation retained significance with the rate of response (odds ratio [OR], 0.48; 95% confidence interval [CI], 0.31 to 0.74; P < .001). Also in multivariate analysis, reduced survival after the start of tamoxifen therapy was observed in the p53-high group (relative hazards rate [RHR], 1.56, 95% CI, 1.17 to 2.10; P = .002). A statistically significant association between p53 levels and decreased tamoxifen response was seen only in the subset of patients whose tumors expressed low levels of ER or PgR (<75 fmol/mg protein). CONCLUSION Measurement of primary tumor p53 levels may be effective in predicting response to tamoxifen therapy in recurrent breast disease. However, more confirming studies on the association between p53 protein accumulation and response to antiestrogen therapy are needed before tumor p53 levels can be used in routine clinical practice.

The impact of CYP2D6-predicted phenotype on tamoxifen treatment outcome in patients with metastatic breast cancer

Background:Cytochrome P450 2D6 (CYP2D6) has a crucial role in the metabolic conversion of tamoxifen into the active metabolite endoxifen. In this cohort study, the effect of CYP2D6-predicted phenotype, defined as the combined effect of CYP2D6 genetic variation and concomitant use of CYP2D6-inhibiting medication, on time to breast cancer progression (TTP) and overall survival (OS) in women who use tamoxifen for metastatic breast cancer (MBC) was examined.Methods:We selected patients treated with tamoxifen (40 mg per day) for hormone receptor-positive MBC from whom a blood sample for pharmacogenetic analysis (CYP2D6*3, *4, *5, *6, *10 and *41) was available. Patient charts (n=102) were reviewed to assess TTP and OS, and to determine whether CYP2D6 inhibitors were prescribed during tamoxifen treatment.Results:OS was significantly shorter in patients with a poor CYP2D6 metaboliser phenotype, compared with extensive metabolisers (HR=2.09; P=0.034; 95% CI: 1.06–4.12). Co-administration of CYP2D6 inhibitors alone was also associated with a worse OS (HR=3.55; P=0.002; 95% CI: 1.59–7.96) and TTP (HR=2.97; P=0.008; 95% CI: 1.33–6.67) compared with patients without CYP2D6 inhibitors.Conclusion:CYP2D6 phenotype is an important predictor of treatment outcome in women who are receiving tamoxifen for MBC. Co-administration of CYP2D6 inhibitors worsens treatment outcome of tamoxifen and should therefore be handled with care.

Prognostic factors in human primary breast cancer: Comparison of c-myc and HER2/neu amplification

Amplification of oncogenes in primary tumours may have prognostic and/or therapeutic significance for patients with breast cancer. We have studied HER2/neu and c-myc amplification together with steroid receptors in human primary breast tumours and related the outcome with (relapse-free) survival. A strong inverse correlation was found between HER2/neu amplification and the presence of oestrogen and progesterone receptors. Actuarial 5-years survival showed that breast cancer patients with c-myc amplification in their primary tumours experience a shorter relapse-free survival, especially in node-negative and in receptor-positive tumours, whereas HER2/neu amplification may be of prognostic value for overall survival in receptor-negative tumours. Overall, in our hands, c-myc amplification appeared to be a more potent prognosticator than HER2/neu amplification in human primary breast cancer.

Infrequent CDKN2 (MTS1/p16) gene alterations in human primary breast cancer

Changes which lead to excessive cyclin production or to loss of cell cycle inhibition by proteins such as p16/MTS1 may release breast tumour cells from the constraints of cell division. In order to establish the frequency of MTS1/p16 gene alteration and its relation with genetic damage to the p53 and cyclin D1 genes, we have studied these gene abnormalities in 164 human primary breast cancers and in six breast cancer cell lines. Two breast cancer cell lines and one primary tumour showed a homozygous deletion of exon 2 of the MTS1 gene. Using single-strand conformation polymorphism and subsequent sequencing analysis, one tumour showed an alteration at codon 67 (CCC-->CTC; Pro to Leu). Another tumour showed a mutation at codon 98 (without amino acid change) with an additional polymorphism at codon 140. This polymorphism was also found in 13 other tumour samples, but has no effect on (disease-free) survival. From these data we conclude that the occurrence of CDKN2 (p16/MTS1) mutation in primary breast cancer is a rare event and is not likely to be involved in human breast tumour carcinogenesis and progression.

Survival and contralateral breast cancer in CHEK2 1100delC breast cancer patients: impact of adjuvant chemotherapy

Background:We assessed the sensitivity to adjuvant chemotherapy in cell cycle checkpoint kinase 2 (CHEK2) vs non-CHEK2 breast cancer patients by comparing the contralateral breast cancer incidence and distant disease-free and breast cancer-specific survival between both groups, stratified for adjuvant chemotherapy.Methods:One Dutch hereditary non-BRCA1/2 breast cancer patient cohort (n=1220) and two Dutch cohorts unselected for family history (n=1014 and n=2488, respectively) were genotyped for CHEK2 1100delC. Hazard ratios for contralateral breast cancer, distant disease-free and breast cancer-specific death for mutation carriers vs noncarriers were calculated using the Cox proportional hazard method, stratified for adjuvant chemotherapy.Results:The CHEK2 mutation carriers (n=193) had an increased incidence of contralateral breast cancer (multivariate hazard ratio 3.97, 95% confidence interval 2.59–6.07). Distant disease-free and breast cancer-specific survival were similar in the first 6 years in mutation carriers compared with noncarriers, but diverted as of 6 years after breast cancer diagnosis (multivariate hazard ratios and 95% confidence intervals 2.65 (1.79–3.93) and 2.05 (1.41–2.99), respectively). No significant interaction between CHEK2 and adjuvant chemotherapy was observed.Conclusions:The CHEK2 1100delC-associated breast cancer is associated with a higher contralateral breast cancer rate as well as worse survival measures beyond 6 years after diagnosis. No differential sensitivity to adjuvant chemotherapy was observed in CHEK2 patients.