Transcriptional expression of 8 genes predicts pathological response to first-line docetaxel + trastuzumab-based neoadjuvant chemotherapy.

BACKGROUND: Overexpression of HER2 is observed in 20 to 30% of breast carcinomas. The use of trastuzumab has improved the treatment of these patients, especially when it is associated with docetaxel. To optimize the use of this treatment, it seems important to select putative complete responders before treatment administration. METHODS: In this study, we analyzed by quantitative PCR the expression of 28 genes in HER2-overexpressing tumors treated with trastuzumab + docetaxel-based chemotherapy. We then correlated their expression profile with those of trastuzumab-sensitive and resistant cell lines to classify tumors as having a sensitive (pCR) or resistant (non-pCR) profile. Finally, we used public datasets from the GEO website to validate the reduced gene-expression profile obtained. RESULTS: We identified an 8-gene-expression combination that predicted the response to treatment with an accuracy of 76%. Based on public microarray data, we showed that the expression profile was specific to first-line trastuzumab + docetaxel-based treatment with an accuracy of 85%. CONCLUSIONS: Our results showed that by profiling the expression of 8 genes it was possible to predict the response to first-line trastuzumab + docetaxel-based chemotherapy. The use of cancer cell lines as the reference allowed a proper fit with the specificity of different tissues, such as lung or gastric cancers, which could also be eligible to concomitant HER2 inhibition by treatment with trastuzumab or tyrosine kinase inhibitors and docetaxel.

Transcriptional regulation of the survivin gene.

Survivin, a small member of the inhibitors of the apoptosis protein family, is highly deregulated in cancer. It is weakly expressed in normal tissues but very strongly expressed in malignant lesions. Survivin is involved in cell-cycle progression, especially in the G2/M transition, and has anti-apoptotic activity, which correlates with its strong expression in cases with a poor cancer treatment response and poor outcomes. Several therapies that target the survivin transcript or protein are currently being tested in clinical trials. However, focusing new therapies on the origins of survivin overexpression and targeting these upstream deregulations could be more effective. For this reason, it seems important to make an inventory of the transcriptional (de)regulation of survivin. This review will gather the important points concerning the regulation of survivin mRNA expression: structure of the survivin promoter, epigenetic modifications and genetic abnormalities, transcription factors, and signalling pathways that affect survivin mRNA expression.

Survivin-3B potentiates immune escape in cancer but also inhibits the toxicity of cancer chemotherapy.

Dysregulation in patterns of alternative RNA splicing in cancer cells is emerging as a significant factor in cancer pathophysiology. In this study, we investigated the little known alternative splice isoform survivin-3B (S-3B) that is overexpressed in a tumor-specific manner. Ectopic overexpression of S-3B drove tumorigenesis by facilitating immune escape in a manner associated with resistance to immune cell toxicity. This resistance was mediated by interaction of S-3B with procaspase-8, inhibiting death-inducing signaling complex formation in response to Fas/Fas ligand interaction. We found that S-3B overexpression also mediated resistance to cancer chemotherapy, in this case through interactions with procaspase-6. S-3B binding to procaspase-6 inhibited its activation despite mitochondrial depolarization and caspase-3 activation. When combined with chemotherapy, S-3B targeting in vivo elicited a nearly eradication of tumors. Mechanistic investigations identified a previously unrecognized 7-amino acid region as responsible for the procancerous properties of survivin proteins. Taken together, our results defined S-3B as an important functional actor in tumor formation and treatment resistance.

Only missense mutations affecting the DNA binding domain of p53 influence outcomes in patients with breast carcinoma.

The presence of a TP53 gene mutation can influence tumour response to some treatments, especially in breast cancer. In this study, we analysed p53 mRNA expression, LOH at 17p13 and TP53 mutations from exons 2 to 11 in 206 patients with breast carcinoma and correlated the results with disease-free and overall survival. The observed mutations were classified according to their type and location in the three protein domains (transactivation domain, DNA binding domain, oligomerization domain) and correlated with disease-free and overall survival. In our population, neither p53 mRNA expression nor LOH correlated with outcome. Concerning TP53 mutations, 27% of tumours were mutated (53/197) and the presence of a mutation in the TP53 gene was associated with worse overall survival (p = 0.0026) but not with disease-free survival (p = 0.0697), with median survival of 80 months and 78 months, respectively. When alterations were segregated into mutation categories and locations, and related to survival, tumours harbouring mutations other than missense mutations in the DNA binding domain of P53 had the same survival profiles as wild-type tumours. Concerning missense mutations in the DNA binding domain, median disease-free and overall survival was 23 months and 35 months, respectively (p = 0.0021 and p<0.0001, respectively), compared with 78 and 80 months in mutated tumours overall. This work shows that disease-free and overall survival in patients with a frameshift mutation of TP53 or missense mutation in the oligomerization domain are the same as those in wild-type TP53 patients.

Regulation of monocarboxylate transporter MCT1 expression by p53 mediates inward and outward lactate fluxes in tumors.

The monocarboxylate transporter (MCT) family member MCT1 can transport lactate into and out of tumor cells. Whereas most oxidative cancer cells import lactate through MCT1 to fuel mitochondrial respiration, the role of MCT1 in glycolysis-derived lactate efflux remains less clear. In this study, we identified a direct link between p53 function and MCT1 expression. Under hypoxic conditions, p53 loss promoted MCT1 expression and lactate export produced by elevated glycolytic flux, both in vitro and in vivo. p53 interacted directly with the MCT1 gene promoter and altered MCT1 mRNA stabilization. In hypoxic p53(-/-) tumor cells, NF-κB further supported expression of MCT1 to elevate its levels. Following glucose deprivation, upregulated MCT1 in p53(-/-) cells promoted lactate import and favored cell proliferation by fuelling mitochondrial respiration. We also found that MCT1 expression was increased in human breast tumors harboring p53 mutations and coincident features of hypoxia, with higher MCT1 levels associated with poorer clinical outcomes. Together, our findings identify MCT1 as a target for p53 repression and they suggest that MCT1 elevation in p53-deficient tumors allows them to adapt to metabolic needs by facilitating lactate export or import depending on the glucose availability.

Apoptosis gene signature of Survivin and its splice variant expression in breast carcinoma.

Survivin, an anti-apoptotic protein, was described as strongly expressed in human cancers including breast cancer. However, little is known about the association between Survivin variants (Survivin-2B, Survivin-ΔEx3, Survivin-3B, and Survivin-2α) and the other apoptotic-related genes. In this study, we analyzed the apoptosis gene signature of Survivin and its variant expression in breast cancer. Human Apoptosis Gene Arrays were used to screen genes that could be associated with Survivin variants. Expression of the five transcripts was measured by RT-PCR in 135 breast carcinomas and Cox survival analysis was analyzed according to the patient outcome. Significant associations between Survivin transcripts and apoptotic genes were found. Interestingly, Survivin-3B variant showed major inverse correlations with pro-apoptotic genes. In addition, in vitro results indicated that overexpression of Survivin-3B strongly inhibits 5-fluorouracil/epirubicin/cyclophosphamide-induced apoptosis in breast tumor cell lines. In breast carcinomas, uni- and multivariate analysis showed patients with high level of Survivin-3B expression had a shorter overall (P=0.030 and P=0.042 respectively), and disease-free (P=0.024 and P=0.009) survival. Our data suggest that Survivin-3B contributes to cell survival through the anti-apoptotic pathway and that its expression level could be an important factor in determining therapeutic strategies for breast carcinoma.

A short caspase-3 isoform inhibits chemotherapy-induced apoptosis by blocking apoptosome assembly.

Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by caspase-3s remains unknown. Here we show that exogenous caspase-3 sensitizes MCF-7 and HBL100 breast cancers cells to chemotherapeutic treatments such as etoposide and methotrexate whereas co-transfection with caspase-3s strongly inhibits etoposide and methotrexate-induced apoptosis underlying thus the anti-apoptotic role of caspase-3s. In caspase-3 transfected cells, lamin-A and α-fodrin were cleaved when caspase-3 was activated by etoposide or methotrexate. When caspase-3s was co-transfected, this cleavage was strongly reduced. Depletion of caspase-3 by RNA interference in HBL100 containing endogenous caspase-3s caused reduction in etoposide and methotrexate-induced apoptosis, whereas the depletion of caspase-3s sensitized cells to chemotherapy. In the presence of caspase-3s, a lack of interaction between caspase-3 and caspase-9 was observed. Immunoprecipitation assays showed that caspase-3s binds the pro-forms of caspase-3. This result suggested that the absence of interaction with caspase-9 when both variants of caspase-3 are present contribute to block the apoptosome assembly and inhibit apoptosis. These data support that caspases-3s negatively interferes with caspase-3 activation and apoptosis in breast cancer, and that it can play key roles in the modulation of response to chemotherapeutic treatments.

Variations in gene expression and response to neoadjuvant chemotherapy in breast carcinoma.

In this study, we performed a screening of 266 gene expressions in breast carcinomas and carried out correlations with histological response to either FEC-100 (fluorouracil-epirubicin-cyclophosphamide; n = 16) or Tax-Epi (docetaxel-epirubicin; n = 12) treatment. Gene expression in biopsies obtained before and after one course of chemotherapy was analyzed. Expression of specific genes was significantly changed after one course of chemotherapy, particularly for Tax-Epi treatment. Comparison with histological response for both treatments revealed that only good responders exhibited changes in gene-expression profile. These results agree that expression of different genes changes in response to anthracycline-based neoadjuvant chemotherapy and show, for the first time, that response to docetaxel-based treatment implied also changes in gene-expression profile.

Predictive value of survivin alternative transcript expression in locally advanced breast cancer patients treated with neoadjuvant chemotherapy.

Survivin, a member of the apoptosis inhibitor protein family, is expressed in numerous human tumours, and its expression is described as a negative prognostic marker. Four alternative splice variants (survivin-DeltaEx3, survivin-3B, survivin-2B and survivin-2alpha) have been described. To date, little is known about the prognostic or predictive role of all five survivin transcripts in breast cancer. In this study, we analysed, by means of real-time quantitative PCR, the five survivin transcripts in a population of 60 breast carcinoma patients treated with 5-fluorouracil + epirubicin + cyclophosphamide (FEC, n=32) or with docetaxel + epirubicin (Tax-Epi, n=28). For each patient, samples were obtained before and after one course of chemotherapy. Before treatment, the ratio of survivin-2alpha was significantly higher in resistant than in sensitive tumours treated by the FEC regimen (p=0.0161), while the ratio of survivin-DeltaEx3 was higher in sensitive than in resistant samples treated with Tax-Epi (p=0.0234). After one course of chemotherapy, expression of survivin-3B was significantly associated with resistance (p=0.0448) in the FEC treatment group, and the ratios of survivin-DeltaEx3 (p=0.0071) and survivin-2B (p=0.0380) were significantly higher in sensitive than in resistant tumours in the Tax-Epi treatment group. Notably, increased expression and ratio of survivin-3B after one course of Tax-Epi was associated with reduced disease-free survival (p=0.0299 and 0.0277, respectively) and with reduced overall survival (p=0.0145 and <0.0001, respectively) of the patients. These results indicate that an imbalance in the alternative transcript ratios may make the cells resistant or sensitive to apoptosis. They also demonstrate for the first time that alternative survivin transcript expression levels may be predictive markers in FEC and Tax-Epi treatment in breast carcinoma.

The expression of BIRC5 is correlated with loss of specific chromosomal regions in breast carcinomas.

Expression of BIRC5 (survivin), a member of the inhibitor of apoptosis protein (IAP) family, is elevated in fetal tissues and in various human cancers. Mechanisms up-regulating BIRC5 in cancer are poorly understood. Here, we show that overexpression of BIRC5 induces a high proliferation level in MCF-7 breast tumor cells. In a population of 191 breast carcinomas, BIRC5 expression is not affected by BIRC5 promoter polymorphism at -31, or BIRC5 gene copy number. However, a significant correlation was found between expression of demethylase (dMTase) and expression of BIRC5. In addition, among 13 chromosomal regions tested for allelic loss [loss of heterozygosity (LOH)], two regions close to D3S1478 and D6S264 were related to BIRC5 expression. In tumors with LOH at D3S1478 and/or D6S264, BIRC5 expression was significantly increased. These regions have been suggested to harbor tumor suppressor genes and/or common fragile sites that may play a role in increasing genetic instability. These results suggest that genes located near D3S1478 and D6S264 might work by inhibiting, directly or indirectly, BIRC5 expression and thus their loss leads to its up-regulation. In addition, BIRC5 expression may induce breast tumor proliferation by promoting genetic instability. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Patterns of loss of heterozygosity in breast carcinoma during neoadjuvant chemotherapy.

There is evidence indicating that resistance to some chemotherapy drugs is related to enhanced repair of DNA lesions. Microsatellite instability (MSI) and loss of heterozy-gosity (LOH) reflect genetic instability and are associated with specific DNA repair pathways. Despite the strong implication of genetic instability in breast cancer its association with chemotherapy is unknown. Thus, we analyzed microsatellite alterations with 12 markers in locally advanced breast carcinomas in relation to neoadjuvant epirubicin-cyclophosphamide-containing chemotherapy (FEC-100) and compared it to a docetaxol-based (Tax-Epi) regimen. Samples were obtained before, during and after treatments. In pre-treated samples, MSI was detected only in 2 cases (7%) whereas LOH was found in 23 of the 34 (68%) carcinomas including 10 belonging to the FEC-100 group and 13 to Tax-Epi one. LOH frequency decreased from the first course of both regimens, but differences between the patterns of LOH during treatment were found. Persistent LOH was more frequent in FEC-100 group (71% vs. 41%) that was detected only in biopsies belonging to non-responder patients. Persistent LOH were clustered at particular loci located at regions containing common fragile sites (FHIT and FRA6E). Analysis of baseline LOH with 6 markers located at 3p indicates discontinuous patterns reflecting double-strand break (DSB) lesions. These results agree with a drug-dependent link between genetic instability and chemoresistance and show that FEC-100 treatment is associated with DSB accumulation manifested as LOH in tumor cells resistant to chemotherapy in breast carcinoma.

Overexpression of caspase-3s splice variant in locally advanced breast carcinoma is associated with poor response to neoadjuvant chemotherapy.

PURPOSE: CASP-3 gene gives rise, by alternative splicing to a caspase-3s variant, to the antagonist apoptotic property of caspase-3. Deregulation of splicing in tumor cells favoring the expression of antiapoptotic variants has been reported to contribute to both tumorigenesis and chemoresistance. Thus, we investigated the role of caspase-3 and its splice variant in breast cancer cells. EXPERIMENTAL DESIGN: Breast tumor cell lines deficient (MCF-7) and proficient (HBL100) for CASP-3 gene were transfected with each transcript and were characterized for their apoptotic response to cyclophosphamide. Expression of the two transcripts were measured by reverse transcription-PCR in 130 breast carcinomas, including 90 locally advanced tumors treated with neoadjuvant chemotherapy containing cyclophosphamide, epirubicine, and 5-fluorouracil. RESULTS: Overexpression of caspase-3s variant in caspase-3-transfected cell lines significantly inhibits apoptosis induced by cyclophosphamide (P < 0.0001 for both cell lines). In breast tissues, only caspase-3 levels were higher in carcinomas than in corresponding adjacent normal tissues (P = 0.0396). Locally advanced carcinomas with high levels of caspase-3 (P < 0.0001) and weak levels of caspase-3s (P = 0.0248) were more sensitive to treatment. Therefore, increase in caspase-3s/caspase3 ratio expression was significantly associated with chemoresistance (P = 0.01). Logistic univariate and multivariate analyses realized according to pathologic response confirm that increased caspase-3s expression was indicative of chemoresistance (P = 0.012 and P = 0.026, respectively). CONCLUSIONS: The results agree with an antagonist function between the two transcripts of caspase-3 and show that their ratio of expression levels may define a subset of locally advanced breast cancer patients who are more likely to benefit from neoadjuvant cyclophosphamide-containing chemotherapy.

Distinct expression of Survivin splice variants in breast carcinomas.

Survivin, a member of the inhibitor apoptosis family, is expressed in several human tumours, and its expression is regulated by p53. Recently, three alternative splice variants (Survivin-2B, Survivin-deltaEx3 and Survivin-3B), differing in their antiapoptotic properties, were identified. To date, little is known about the expression of all Survivin splice variants in breast cancer, particularly the recently identified Survivin-3B variant. In this study, we show that all Survivin transcripts were expressed in breast tumour cell lines and breast carcinomas, with a very weak expression detected in adjacent normal tissue. Frequency of proapoptotic Survivin-2B was significantly higher in small tumour size (p=0.026) and was inversely associated with axillary node positive carcinomas (p=0.004). In contrast, Survivin-3B was more frequent in high-grade carcinomas (p=0.004). Correlation with p53 status revealed that Survivin-3B was significantly more frequent in carcinomas with p53 gene mutation (p=0.036). After neoadjuvant chemotherapy, a significant reduction in the percentage of expressing Survivin (p=0.016) and Survivin-2B (p=0.027) was observed, while no change was found for Survivin-deltaEx3 and Survivin-3B variants. These results demonstrate for the first time that Survivin variants are differentially expressed in breast cancer according to tumour progression and treatment and suggest that Survivin-3B might act as an antiapoptotic factor in this lesion, with its expression regulated by p53.

Comparative analysis of molecular alterations in fibroadenomas associated or not with breast cancer.

HYPOTHESIS: The cause of breast cancer is linked to many macroscopic events, including benign breast disease. In this study we asked whether molecular changes could discriminate fibroadenoma, which is one of the most common benign breast disease lesions associated or not with breast cancer. DESIGN: Retrospective cohort study. SETTING: Anticancer medical center. SUBJECTS: Archival tissues in 32 cases of fibroadenoma, diagnosed in the same breast as a breast carcinoma, are compared with a control group of 26 cases of fibroadenomas unaffected by breast cancer. MAIN OUTCOME MEASURES: Histological features are characterized in all samples. The epithelial and stromal components are analyzed for a loss of heterozygosity and a microsatellite instability using a polymerase chain reaction-based method with 11 polymorphic microsatellite markers at 7 chromosomal regions frequently altered in breast cancer. The p53 gene mutations were also determined at exons 5 to 9. RESULTS: The frequency of complex fibroadenomas was similar in both groups (P =.42). Only in the case group did we observe proliferative lesions confined in fibroadenomas, including atypical ductal hyperplasia (2 cases), lobular neoplasia (3 cases), or low-grade ductal carcinoma in situ (2 cases). There is no significant morphological difference between the 2 groups. Neither microsatellite alterations nor p53 gene mutations are present in the fibroadenoma components. Loss of heterozygosity is found only in the epithelial component of the 2 ductal carcinomas in situ confined in fibroadenomas. CONCLUSIONS: Genetic alterations, which are most frequently involved in malignant breast carcinomas, are not present in fibroadenomas, regardless of their association with breast cancer or their histological complexity. These findings suggest that fibroadenomas are not associated with breast carcinogenesis.

Analysis of microsatellite instability in acquired drug-resistance human tumor cell lines.

Genomic instability characterized as microsatellite instability (MIN) is associated with loss of DNA mismatch repair (MMR) protein. Several studies have shown that loss of DNA MMR protein confers resistance to some interacting DNA chemotherapeutic drugs, but also that exposure of MMR-proficient cells to these drugs can result in loss of MMR protein accompanied by induction of MIN. Such associations were mainly reported for cisplatin, but scarce data are available for doxorubicin (a DNA interacting agent), and nothing is known about vinblastine (an antitubulin agent). Thus, in this study we have analyzed MIN frequency in different type of human tumor cell lines characterized by their MMR protein status and resistant to doxorubicin or to vinblastine. Relationship between MIN occurrence and drug resistance was firstly verified in cisplatin resistant cells, and showed a MIN enrichment (33%) only in the MMR-deficient cells. In order to determine whether treatment of MMR-proficient cells with doxorubicin might lead to induction of MIN, we analyzed two different MMR-proficient cell lines. Variations of MIN frequency were found with either high levels of MIN (66%) or no MIN at all (0%). Effect of vinblastine was analyzed according to the MMR status in two different MMR-proficient and -deficient cells. No major change in MIN frequency was found either in the MMR-proficient (0%) or -deficient (9%) cells. Our results demonstrate that MIN occurs only in tumor cells resistant to cisplatin or doxorubicin, thus supporting earlier findings reporting such associations only with drugs interacting with DNA. Moreover, the data show that MIN does not appear in all tumor cell lines, suggesting that induction of MIN in relation to MMR status is a complex phenomenon which does not only depend on the drug considered (interacting or not with DNA), but also on the tumor cell variant.

Human colon cancer cells surviving high doses of cisplatin or oxaliplatin in vitro are not defective in DNA mismatch repair proteins.

PURPOSE: Alterations in the DNA mismatch repair (MMR) proteins have been associated with an increased resistance of many cancer cell lines to cisplatin. The aim of this work was to investigate whether defects in DNA MMR proteins are involved in the survival of human colorectal cancer cells in the presence of high concentrations of cisplatin and oxaliplatin, a diaminocyclohexane (DACH) platinum compound whose adducts are not recognized by the MMR system. METHODS: Six unselected human colon cancer cell lines (HT29, HCT15, HCT116, Caco2, SW480 and SW620) were treated with a single 3-h exposure to cisplatin or oxaliplatin at suprapharmacological concentrations, ranging from 50 to 200 microg/ml. The microsatellite stability and the expression of MMR proteins in the parental cell lines and in the drug-selected subpopulations were studied. RESULTS: Most cells underwent apoptosis in the days following the cisplatin or oxaliplatin treatment, but some colonies expanded 3 to 4 weeks after, suggesting the presence of innately resistant cells in the six parental cell lines. Microsatellite instability (MIN), which reflects genetic defects in the DNA MMR system, was detected only in the HCT116 parental cell line and its drug-selected counterparts, due to a known mutation in the hMLH1 gene. No acquired MIN was observed in the other cisplatin-selected sublines derived from the HT29, HCT15, Caco2, SW480 or SW620 parental cells. In the same way, Western blot analysis showed that expression of the DNA MMR proteins hMLH1, hPMS1, hPMS2, hMSH2 and hMSH6 did not differ between the parental and the drug-surviving cells. CONCLUSIONS: These results indicate that high-level resistance of human colon cancer cells to high doses of cisplatin and oxaliplatin does not seem to be related to acquired defects in the DNA MMR proteins.

Breast carcinoma metastasis within granulosa cell tumor of the ovary: morphologic, immunohistologic, and molecular analyses of the two different tumor cell populations.

Gynecologic metastasis of breast carcinoma is not an infrequent event, but metastases within another tumor is very rare. We report a case of unilateral ovarian tumor arising in a 63-year-old woman receiving tamoxifen therapy with a past history of breast carcinoma. The microscopic appearance was principally that of a granulosa cell tumor, but the presence of atypical cells closely admixed within the classical areas was reminiscent of metastasis from breast carcinoma. The diagnosis of this first reported case of breast carcinoma metastasis within granulosa cell tumor was supported by immunohistologic analysis. The diagnosis of tumor-to-tumor metastasis was also confirmed by molecular study using microdissections of samples from the initial breast tumor and from the subsequent ovarian tumor. When compared with normal tissue, carcinomatous cells in the breast tissue exhibited genomic abnormality at the same locus as the metastatic cells in the ovary. In contrast, granulosa cell tumor areas did not show any loss of heterozygosity or instability for the microsatellites analyzed.