Chk2 splice variants express a dominant-negative effect on the wild-type Chk2 kinase activity.

While the majority of RNA transcripts from protein-encoding genes in the human genome are subject to physiological splicing, pathological splicing is increasingly reported in cancer tissue. Previously, we identified >90 different splice variants of Chk2, a gene encoding a serine/threonine kinase propagating the DNA damage signal by phosphorylating and activating several downstream substrates like p53, Cdc25A, and Cdc25C involved in cell cycle arrest and apoptosis. While alternative splice forms of other genes have been reported to exert a dominant-negative effect on the wild-type molecules, the function of Chk2 splice protein variants is still unclear. Here we evaluated the function of four Chk2 splice proteins for which mRNA splice variants were identified in human breast carcinomas. These splice variants were stably expressed as nuclear proteins. Two splice forms (Chk2Delta4 and Chk2del(2-3)) expressed kinase activity while variants Chk2Delta11 and Chk2isoI were essentially kinase inactive. Independent of intrinsic kinase activity, each splice variant impaired wild-type Chk2 activity through heterodimerization. Based on our findings, we suggest alternative splicing as a possible novel mechanism for repression of the Chk2 wild-type function.

Identification and characterization of retinoblastoma gene mutations disturbing apoptosis in human breast cancers.

BACKGROUND: The tumor suppressor pRb plays a key role regulating cell cycle arrest, and disturbances in the RB1 gene have been reported in different cancer forms. However, the literature reports contradictory findings with respect to a pro--versus anti--apoptotic role of pRb, and the consequence of alterations in RB1 to chemotherapy sensitivity remains unclear. This study is part of a project investigating alterations in pivotal genes as predictive factors to chemotherapy sensitivity in breast cancer. RESULTS: Analyzing 73 locally advanced (stage III) breast cancers, we identified two somatic and one germline single nucleotide changes, each leading to amino acid substitution in the pRb protein (Leu607Ile, Arg698Trp, and Arg621Cys, respectively). This is the first study reporting point mutations affecting RB1 in breast cancer tissue. In addition, MLPA analysis revealed two large multiexon deletions (exons 13 to 27 and exons 21 to 23) with the exons 21-23 deletion occurring in the tumor also harboring the Leu607Ile mutation. Interestingly, Leu607Ile and Arg621Cys point mutations both localize to the spacer region of the pRb protein, a region previously shown to harbor somatic and germline mutations. Multiple sequence alignment across species indicates the spacer to be evolutionary conserved. All three RB1 point mutations encoded nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro. Notably, three out of four tumors harboring RB1 mutations displayed primary resistance to treatment with either 5-FU/mitomycin or doxorubicin while only 14 out of 64 tumors without mutations were resistant (p = 0.046). CONCLUSIONS: Although rare, our findings suggest RB1 mutations to be of pathological importance potentially affecting sensitivity to mitomycin/anthracycline treatment in breast cancer.

The novel p21 polymorphism p21G251A is associated with locally advanced breast cancer.

PURPOSE: p21 is a main effector of growth arrest induced by p53. In addition, a second transcript from the same gene (p21B) has been linked to apoptosis. We previously analyzed p21 status in breast cancer and reported two novel polymorphisms of the p21 gene. In the present study, we present a larger study designed to explore a possible association between these novel polymorphisms and breast cancer. EXPERIMENTAL DESIGN: The p21/p21B polymorphisms were analyzed in 507 breast cancer patients and 1,017 healthy individuals using cDNA or genomic DNA from tumor and/or blood samples. RESULTS: We detected five polymorphisms of the p21 gene. Three of these polymorphisms are earlier reported by others, whereas two were reported for the first time in a recent study by us. The presence of the A allele of the p21G251A polymorphism was observed more frequently among patients with primary stage III breast cancer (4.5%) compared with stage I and II tumors (1.5%) and healthy female controls (1.4%; P = 0.007, comparing the three groups; P = 0.0049 and P = 0.0057, comparing locally advanced to stage I/II and healthy controls, or to healthy controls alone, respectively). The allele frequencies of the remaining four polymorphisms were evenly distributed among patients and healthy individuals. DISCUSSION: The finding of an association between locally advanced breast cancer and one particular polymorphism of the p21 gene suggests this polymorphism to be related to tumor behavior, including enhanced growth rate. If confirmed in other studies, this may add significant information to our understanding of the biology as well as of the clinical behaviour of locally advanced breast cancers.

Alternative splicing and mutation status of CHEK2 in stage III breast cancer.

The DNA damage checkpoint kinase, CHK2, promotes growth arrest or apoptosis through phosphorylating targets such as Cdc25A, Cdc25C, BRCA1, and p53. Both germline and somatic loss-of-function CHEK2 mutations occur in human tumours, the former linked to the Li-Fraumeni syndrome, and the latter found in diverse types of sporadic malignancies. Here we examined the status of CHK2 by genetic and immunohistochemical analyses in 53 breast carcinomas previously characterized for TP53 status. We identified two CHEK2 mutants, 470T>C (Ile157Thr), and a novel mutation, 1368insA leading to a premature stop codon in exon 13. The truncated protein encoded by CHEK2 carrying the 1368insA was stable yet mislocalized to the cytoplasm in tumour sections and when ectopically expressed in cultured cells. Unexpectedly, we found CHEK2 to be subject to extensive alternative splicing, with some 90 splice variants detected in our tumour series. While all cancers expressed normal-length CHEK2 mRNA together with the spliced transcripts, we demonstrate and/or predict some of these splice variants to lack CHK2 function and/or localize aberrantly. We conclude that cytoplasmic sequestration may represent a novel mechanism to disable CHK2, and propose to further explore the significance of the complex splicing patterns of this tumour suppressor gene in oncogenesis.

Genetic and epigenetic changes in p21 and p21B do not correlate with resistance to doxorubicin or mitomycin and 5-fluorouracil in locally advanced breast cancer.

PURPOSE: The cyclin-dependent kinase inhibitor p21 acts as a main executor of p53-induced growth arrest. Recently, a second transcript, p21B, was found to code for a protein expressing proapoptotic activity. We investigated p21 and p21B for mutations and epigenetic silencing in locally advanced breast cancers treated with doxorubicin or 5-fluorouracil/mitomycin and correlated our findings with treatment response and TP53 status. EXPERIMENTAL DESIGN: We used reverse transcription-PCR to analyze p21/p21B mutation status in 73 breast cancer samples. The p21 promoter region was sequenced and analyzed for hypermethylations by methylation-specific PCR. In addition, a selection of patients were analyzed for mutations in the p21B promoter. RESULTS: The p21 gene was neither mutated nor silenced by promoter hypermethylation in any of the tumors examined. One patient harbored a novel p21 splice variant in addition to the wild-type transcript. We observed two base substitutions in the p21 transcript, C93A and G251A, each affecting six patients (8.2%). The G251A variant had not been reported previously. In 12 patients (16.4%), we observed a novel base substitution, T35C, in p21B. All three base substitutions were observed in lymphocyte DNA and therefore considered polymorphisms. The polymorphisms did not correlate with p21 staining index, treatment response to doxorubicin or 5-fluorouracil/mitomycin, or TP53 status. CONCLUSIONS: Our findings do not suggest that genetic or epigenetic disturbances in p21 or p21B cause resistance to doxorubicin or mitomycin/5-fluorouracil in breast cancer. Future studies should assess potential associations between these novel polymorphisms and breast cancer risk.

Concomitant inactivation of the p53- and pRB- functional pathways predicts resistance to DNA damaging drugs in breast cancer in vivo.

Chemoresistance is the main obstacle to cancer cure. Contrasting studies focusing on single gene mutations, we hypothesize chemoresistance to be due to inactivation of key pathways affecting cellular mechanisms such as apoptosis, senescence, or DNA repair. In support of this hypothesis, we have previously shown inactivation of either TP53 or its key activators CHK2 and ATM to predict resistance to DNA damaging drugs in breast cancer better than TP53 mutations alone. Further, we hypothesized that redundant pathway(s) may compensate for loss of p53-pathway signaling and that these are inactivated as well in resistant tumour cells. Here, we assessed genetic alterations of the retinoblastoma gene (RB1) and its key regulators: Cyclin D and E as well as their inhibitors p16 and p27. In an exploratory cohort of 69 patients selected from two prospective studies treated with either doxorubicin monotherapy or 5-FU and mitomycin for locally advanced breast cancers, we found defects in the pRB-pathway to be associated with therapy resistance (p-values ranging from 0.001 to 0.094, depending on the cut-off value applied to p27 expression levels). Although statistically weaker, we observed confirmatory associations in a validation cohort from another prospective study (n = 107 patients treated with neoadjuvant epirubicin monotherapy; p-values ranging from 7.0 × 10(-4) to 0.001 in the combined data sets). Importantly, inactivation of the p53-and the pRB-pathways in concert predicted resistance to therapy more strongly than each of the two pathways assessed individually (exploratory cohort: p-values ranging from 3.9 × 10(-6) to 7.5 × 10(-3) depending on cut-off values applied to ATM and p27 mRNA expression levels). Again, similar findings were confirmed in the validation cohort, with p-values ranging from 6.0 × 10(-7) to 6.5 × 10(-5) in the combined data sets. Our findings strongly indicate that concomitant inactivation of the p53- and pRB- pathways predict resistance towards anthracyclines and mitomycin in breast cancer in vivo.

Different adipose depots: their role in the development of metabolic syndrome and mitochondrial response to hypolipidemic agents.

Adipose tissue metabolism is closely linked to insulin resistance, and differential fat distributions are associated with disorders like hypertension, diabetes, and cardiovascular disease. Adipose tissues vary in their impact on metabolic risk due to diverse gene expression profiles, leading to differences in lipolysis and in the production and release of adipokines and cytokines, thereby affecting the function of other tissues. In this paper, the roles of the various adipose tissues in obesity are summarized, with particular focus on mitochondrial function. In addition, we discuss how a functionally mitochondrial-targeted compound, the modified fatty acid tetradecylthioacetic acid (TTA), can influence mitochondrial function and decrease the size of specific fat depots.

Tissue-specific effects of bariatric surgery including mitochondrial function.

A better understanding of the molecular links between obesity and disease is potentially of great benefit for society. In this paper we discuss proposed mechanisms whereby bariatric surgery improves metabolic health, including acute effects on glucose metabolism and long-term effects on metabolic tissues (adipose tissue, skeletal muscle, and liver) and mitochondrial function. More short-term randomized controlled trials should be performed that include simultaneous measurement of metabolic parameters in different tissues, such as tissue gene expression, protein profile, and lipid content. By directly comparing different surgical procedures using a wider array of metabolic parameters, one may further unravel the mechanisms of aberrant metabolic regulation in obesity and related disorders.

The MDM2 promoter SNP285C/309G haplotype diminishes Sp1 transcription factor binding and reduces risk for breast and ovarian cancer in Caucasians.

MDM2 plays a key role in modulating p53 function. The MDM2 SNP309T > G promoter polymorphism enhances Sp1 binding and has been linked to cancer risk and young age at diagnosis although with conflicting evidence. We report a second MDM2 promoter polymorphism, SNP285G > C, residing on the SNP309G allele. SNP285C occurs in Caucasians only, where 7.7% (95% CI 7.6%-7.8%) of healthy individuals carry the SNP285C/309G haplotype. In vitro analyses reveals that SNP309G enhances but SNP285C strongly reduces Sp1 promoter binding. Comparing MDM2 promoter status among different cohorts of ovarian (n = 1993) and breast (n = 1973) cancer patients versus healthy controls (n = 3646), SNP285C reduced the risk of both ovarian (OR 0.74; CI 0.58-0.94) and breast cancer (OR 0.79; CI 0.62-1.00) among SNP309G carriers.

Mutations and polymorphisms of the p21B transcript in breast cancer.

p21(WAF1/CIP1), transcribed from the CDKN1A locus, plays a key role executing p53-induced growth arrest. The recent discovery that an alternative transcript, p21B, induces apoptosis, suggests an additional important function of this gene. Here, we report p21 and p21B mutation status in large cohorts of breast cancers and compare distributions of p21B polymorphisms in cancer patients to healthy controls. In 521 breast tumor samples analyzed, only one point mutation affecting the p21B protein was observed. No mutations were found when screening a panel of 20 established cell lines. A novel polymorphism, p21B(G128T) was identified. Haplotype analysis revealed no association between this variant and the previously identified p21B polymorphism p21B(T35C) or any of the known p21(WAF1/CIP1) polymorphisms. As previously reported for p21B(T35C), distribution of p21B(G128T) was similar among breast cancer patients and healthy controls (n = 691 and 1,015; incidence 6.1 vs. 4.8%; p = 0.273, respectively). No association between p21B(G128T) or p21B(T35C) and response to chemotherapy with an anthracycline-containing regimen or paclitaxel was recorded. Our findings do not suggest mutations or polymorphisms of p21B to play a major role with respect to either breast cancer risk or sensitivity towards chemotherapy.