Calcitriol supplementation effects on Ki67 expression and transcriptional profile of breast cancer specimens from post-menopausal patients.

BACKGROUND & AIMS: High concentration of 1,25(OH)2D3 (50-100 nM), which cause hypercalcemia in vivo, induce the hormone transcriptional targets and exert antiproliferative effects in cultured breast cancer lineages, however, no studies investigated whether these effects might be reproduced in tumor specimens in vivo. Our aim was to evaluate the effects of calcitriol supplementation on the proliferative index (Ki67 expression) and gene expression profile of post-menopausal breast cancer samples. METHODS & RESULTS: Tumor samples were collected from 33 patients, most of whom (87.5%) presenting 25(OH)D3 insufficiency, before and after a short term calcitriol supplementation (0.50 µg/day PO, for 30 days). Tumor dimension remained stable in ultrasound evaluations. A slight reduction in Ki67 immunoexpression was detected, however in only 10/32 post-calcitriol samples an expressively low proliferative index [Ln (%Ki67+) < 1] was achieved. Gene expression from 15 matched pre/post-supplementation samples was analyzed by microarray (U133 Plus 2.0 GeneChip, Affymetrix) and 15 genes were over-expressed in post-supplementation tumors, including FOS and EGR1, which were previously shown to be regulated by vitamin D. However, these results were not confirmed in another four breast cancer samples. CONCLUSIONS: Calcitriol supplementation is neither sufficient to expressively elicit an antiproliferative response nor to induce the hormone transcriptional signaling pathway in breast cancer specimens.

Expressão gênica diferencial do câncer de mama de pacientes pós-menopausadas responsivas e não-responsivas ao efeito antiproliferativo da vitamina D / Breast cancer gene expression profile in post-menopausal patients responsive or non-responsive to the antiproliferative effect of vitamin D

Baixos níveis séricos de 25(OH)D3 e 1,25(OH)2D3 (calcitriol) podem estar associados à incidência e prognóstico do câncer de mama. Além disso, vários estudos indicam que a vitamina D tenha um efeito antiproliferativo em linhagens celulares de câncer de mama expostas a concentrações supra-fisiológicas de calcitriol (1,25(OH)2D3, 100nM). A suplementação com vitamina D é indicada a mulheres pós-menopausadas para prevenção de osteoporose e observamos previamente que a suplementação de calcitirol a pacientes pós-menopausadas com câncer de mama causa redução do índice proliferativo tumoral. Entretanto, não há estudos até o momento que avaliam o efeito da vitamina D na expressão gênica global in vivo. Incluímos 31 pacientes pós-menopausadas com câncer de mama. Estas pacientes realizaram suplementação com calcitriol (0,5g/dia, dose indicada para prevenção de osteoporose) por um curto período de tempo (mediana de 32 dias). A amostra tumoral foi coletada por ocasião da biópsia (présuplementação) e da ressecção tumoral (pós-suplementação). Os perfis de expressão gênica de 16 pacientes foram analisados a partir de 100ng de RNA total no gene chip U133 Plus 2.0 Affymetrix. Observamos redução na expressão de Ki-67 após a suplementação. Dentre os genes diferencialmente expressos encontram-se EGR1, FOS, DUSP1, MMP12 e RGS1, os quais foram mais expressos em amostras pós-suplementadas. Genes modulados pela vitamina D estão associados à resposta inflamatória e à membrana. Nossos resultados indicam que a suplementação com vitamina D reduz o índice de proliferação tumoral, sendo a mesma envolvida em vias importantes na regulação da resposta inflamatória.

Low 25(OH)2D3 or 1,25(OH)2D3 serum levels may be associated with breast cancer incidence and prognosis. Additionally, the antiproliferative effects of vitamin D are observed in breast cancer cell lines exposed to phamacological doses of calcitriol (1,25(OH)2D3, 100nM). Vitamin D supplementation is indicated for post-menopausal women to prevent osteoporosis and a previous study from our group observed a reduced tumor proliferative index after calcitriol supplementation on post menopausal breast cancer patients. However, there is no study that verifies the effect of vitamin D on gene expression profile in vivo so far. Thirty one post menopausal breast cancer patients were included on our analysis. They were supplemented with calcitriol after tumor biopsy (0.50g/day, indicated dose for osteoporosis prevention) for a short period of time (median 32 days). Tumor samples were collected during biopsy (before supplementation) and breast surgery (after supplementation). Gene expression profile of 16 patients was analyzed using the U133 Plus 2.0 Affymetrix Gene Chips from 100ng of total RNA. After supplementation, a reduced expression of Ki-67 was observed. Among the differentially expressed genes, EGR1, FOS, DUSP1, MMP12 and RGS1 were upregulated after calcitriol supplementation. Differentially expressed genes were involved in inflammatory response or were associated with the membrane. Our results indicate that calcitriol supplementation diminish tumor proliferation index regulating inflammatory pathways.

CYP1A2*1C, CYP2E1*5B, and GSTM1 polymorphisms are predictors of risk and poor outcome in head and neck squamous cell carcinoma patients.

Head and neck squamous cell carcinoma (HNSCC) is associated with environmental factors, especially tobacco and alcohol consumption. Most of the carcinogens present in tobacco smoke are converted into DNA-reactive metabolites by cytochrome P450 (CYPs) enzymes and detoxification of these substances is performed by glutathione S-transferases (GSTs). It has been suggested that genetic alterations, such as polymorphisms, play an important role in tumorigenesis and HNSCC progression. The aim of this study was to investigate CYP1A1, CYP1A2, CYP2E1, GSTM1, and GSTT1 polymorphisms as risk factors in HNSCC and their association with clinicopathologic data. The patients comprised 153 individuals with HNSCC (cases) and 145 with no current or previous diagnosis of cancer (controls). Genotyping of the single nucleotide polymorphisms (SNPs) of the CYP1A1, CYP1A2, and CYP2E1 genes was performed by PCR-RFLP and the GSTM1 and GSTT1 copy number polymorphisms (CNPs) were analyzed by PCR-multiplex. As expected, a significant difference was detected for tobacco and alcohol consumption between cases and controls (P<0.001). It was observed that the CYP1A2*1D (OR=16.24) variant and GSTM1 null alleles (OR=0.02) confer increased risk of HNSCC development (P<0.001). In addition, head and neck cancer alcohol consumers were more frequently associated with the CYP2E1*5B variant allele than control alcohol users (P<0.0001, OR=190.6). The CYP1A2*1C polymorphism was associated with tumor recurrence (log-rank test, P=0.0161). The CYP2E1*5B and GSTM1 null alleles were significantly associated with advanced clinical stages (T3+T4; P=0.022 and P=0.028, respectively). Overall, the findings suggested that the genetic polymorphisms studied are predictors of risk and are also associated with tumor recurrence, since they are important for determining the parameters associated with tumor progression and poor outcomes in HNSCC.