Vasoactive intestinal peptide exerts an osteoinductive effect in human mesenchymal stem cells.

Several neuropeptides present in bone tissues, produced by nerve fibers and bone cells, have been reported to play a role in regulating the fine-tuning of osteoblast and osteoclast functions to maintain bone homeostasis. This study aims to characterize the influence of the neuropeptide vasoactive intestinal peptide (VIP) on the differentiation process of human mesenchymal stem cells (MSCs) into osteoblasts and on their anabolic function. We describe the mRNA and protein expression profile of VIP and its receptors in MSCs as they differentiate into osteoblasts, suggesting the presence of an autocrine signaling pathway in these cells. Our findings reveal that VIP enhances the expression of early osteoblast markers in MSCs under osteogenic differentiation and favors both bone matrix formation and proper cytoskeletal reorganization. Finally, our data suggest that VIP could be exerting a direct modulatory role on the osteoblast to osteoclast signaling by downregulating the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio. These results highlight the potential of VIP as an osteoinductive differentiation factor, emerging as a key molecule in the maintenance of human bone homeostasis.

Genetic 3'UTR variations and clinical factors significantly contribute to survival prediction and clinical response in breast cancer patients.

The study describes a relationship between the 3'UTR variants, clinicopathological parameters and response to chemotherapy. We analyzed 33 germline polymorphisms in 3'UTRs of ADME genes in 305 breast cancer women treated with FAC regime. Clinical endpoints of this study were: overall survival (OS), progression-free survival (PFS), recurrence-free survival (RFS) and overall response defined as treatment failure-free survival (TFFS). The shortened OS was connected with the presence of NR1/2 rs3732359 AA, SLC22A16 rs7756222 CC, as well as SLC22A16 rs9487402 allele G and clinical factors belonging to TNM classification: tumor size >1 cm, nodal involvement and presence of metastases. PFS was related to two polymorphisms PGR rs1824125 GG, PGR rs12224560 CC and SLC22A16 rs7756222 CC as well as preexisting metastases. The RFS was shortened due to the DPYD rs291593 CC, AKR1C3 rs3209896 AG and negative expression of PGR. The presence of ALDH5A1 rs1054899 allele A, lack of pre-chemotherapy surgery and negative status of PGR correlated with worse treatment response. The germline variants commonly present in the population are important factors determining the response to treatment. We observed the effect of the accumulation of genetic and clinical factors on poor survival prognosis and overall treatment response.

Pharmacogenetics of toxicity of 5-fluorouracil, doxorubicin and cyclophosphamide chemotherapy in breast cancer patients.

The differences in patients' response to the same medication, toxicity included, are one of the major problems in breast cancer treatment. Chemotherapy toxicity makes a significant clinical problem due to decreased quality of life, prolongation of treatment and reinforcement of negative emotions associated with therapy. In this study we evaluated the genetic and clinical risk factors of FAC chemotherapy-related toxicities in the group of 324 breast cancer patients. Selected genes and their polymorphisms were involved in FAC drugs transport (ABCB1, ABCC2, ABCG2,SLC22A16), metabolism (ALDH3A1, CBR1, CYP1B1, CYP2C19, DPYD, GSTM1, GSTP1, GSTT1, MTHFR,TYMS), DNA damage recognition, repair and cell cycle control (ATM, ERCC1, ERCC2, TP53, XRCC1). The multifactorial risk models that combine genetic risk modifiers and clinical characteristics were constructed for 12 toxic symptoms. The majority of toxicities was dependent on the modifications in components of more than one pathway of FAC drugs, while the impact level of clinical factors was comparable to the genetic ones. For the carriers of multiple high risk factors the chance of developing given symptom was significantly elevated which proved the factor-dosage effect. We found the strongest associations between concurrent presence of clinical factors - overall and recurrent anemia, nephrotoxicity and early nausea and genetic polymorphisms in genes responsible for DNA repair, drugs metabolism and transport pathways. These results indicate the possibility of selection of the patients with expected high tolerance to FAC treatment and consequently with high chance of chemotherapy completion without the dose reduction, treatment delays and decline in the quality of life.

Genetic polymorphisms and response to 5-fluorouracil, doxorubicin and cyclophosphamide chemotherapy in breast cancer patients.

Clinical resistance to chemotherapy is one of the major problems in breast cancer treatment. In this study we analyzed possible impact of 22 polymorphic variants on the treatment response in 324 breast cancer patients. Selected genes were involved in FAC chemotherapy drugs transport (ABCB1, ABCC2, ABCG2, SLC22A16), metabolism (CYP1B1, CYP2C19, GSTT1, GSTM1, GSTP1, TYMS, MTHFR, DPYD), drug-induced damage repair (ERCC1, ERCC2, XRCC1) and involved in regulation of DNA damage response and cell cycle control (ATM, TP53).Apart from preexisting metastases three polymorphic variants were independent prognostic high risk factors of lack of response to FAC chemotherapy. Our results showed that the response to treatment depended of the variability in genes engaged in drugs' transport (ABCC2 c.-24C>T, ABCB1 p.Ser893Ala/Thr) and in DNA repair machinery (ERCC2 p.Lys751Gln). Furthermore, the growing number of high-risk genotypes was reflected in gradual increase in risk of the non-responsiveness to treatment- from OR 2.68 for presence of two genotypes to OR 9.93 for carriers of all three negative genotypes in the group of all patients. Similar gene-dosage effect was observed in the subgroup of TNBCs. Also, TFFS significantly shortened with the increasing number of high-risk genotypes, with median of 54.4 months for carriers of one variant, to 51.5 and 34.9 months for the carriers of two and three genotypes, respectively.Our results demonstrate that results of cancer treatment are the effect of many clinical and genetic factors. It seems that multifactorial polymorphic models could be a potentially useful tool in personalization of cancer therapies. The novelty in our model is the over representation of triple negative breast cancer (TNBC) patients among the carriers of all unfavorable polymorphic variants. This finding contributes to the elucidation of the mechanisms of drug resistance in this subgroup of breast cancer patients.

A Comparison between CHEK2*1100delC/I157T Mutation Carrier and Noncarrier Breast Cancer Patients: A Clinicopathological Analysis.

OBJECTIVE: The suppressor gene CHEK2 encodes a cell cycle checkpoint kinase, involved in cell cycle regulation, apoptosis and response to DNA damage. The aim of this study was to analyze the differences between CHEK2 mutation carriers (CHEK2*1100delC/I157T) and noncarriers with respect to clinicopathological factors. METHODS: We reviewed the medical records of 100 early breast cancer patients (46 mutation carriers and 54 noncarriers) who were treated with chemotherapy, hormonotherapy or trastuzumab. RESULTS: CHEK2 mutation carriers were older (>65 years) than noncarriers (17 vs. 7%; p = 0.215). Twenty-five (54%) of them had a history of cancer in the family. Gastric cancer in the family history was detected in 11% of mutation carriers and in 2% of noncarriers (p = 0.092). There was a trend for more frequent lymph node metastases in patients without the mutation in comparison to mutation carriers (46 vs. 28%; p = 0.098). Luminal B type breast cancer was detected more often in carriers (39 vs. 20%; p = 0.048). Breast-conserving treatment was also conducted more often in mutation carriers (57 vs. 31%; p = 0.015). Histologic grades G1/G2 were detected more frequently in mutation carriers (82 vs. 70%; p = 0.212). CONCLUSION: Mutation carriers were characterized by older age, a history of gastric cancer in the family, locally advanced disease, lower histologic grade and luminal B type breast cancer.

Genetic polymorphisms and gene-dosage effect in ovarian cancer risk and response to paclitaxel/cisplatin chemotherapy.

BACKGROUND: Ovarian malignancies are often diagnosed in advanced stage and at the same time resistance to treatment, both intrinsic and developed during treatment, is sometimes observed. These facts underscore the need for new markers of ovarian cancer risk, as well as markers of treatment effectiveness. METHODS: In this study we genotyped 225 ovarian cancer patients, 64 breast and ovarian cancer patients and 348 healthy controls. In total, 12 polymorphic variants and 2 deletions in PGR, ABCB1, ABCG2, GSTT1, GSTM1, GSTP1, ATM, TP53 and ATP7B genes were analyzed using ASA-PCR, RFLP-PCR, multiplex-PCR and sequencing. RESULTS: Ten genetic polymorphisms were significantly associated with the risk of developing ovarian carcinoma in at least one of the groups under study. Impact of PGR gene polymorphisms on ovarian cancer risk was specific only for the group of the BRCA1 mutation carriers (in presence of p.Val660Leu variant- OR 2,82; p = 0,010), which confirms the difference in modulation of ovarian cancer risk between sporadic and hereditary malignancies, including the breast-ovarian cancer group (as a cancer-prone group). The analyses showed also the importance of ATP7B gene in ovarian carcinogenesis, both studied variants of which significantly modulated the ovarian cancer risk in all groups excluding the group with BRCA1 mutation. Cumulative risk analysis revealed 3 unfavorable variants that increased significantly the risk of developing ovarian cancer (p.Ile1145 = ABCB1+ p.Asp1853Asn ATM+ p.Ser406Ala ATP7B- OR 7,47; p = 0,002) and significantly modified the progression free survival (PFS) of the patients, and also two favorable genotypes which protected against ovarian cancer (p.Arg952Lys ATP7B+ p.Arg72Pro TP53- OR 0,50; p = 0,008). PFS analysis for carriers of favorable versus unfavorable genotypes emphasized the impact of the regulation of cell cycle (p.Asp1853Asn ATM) and active transport of xenobiotics (p.Ser894Ala/Thr ABCB1) on the risk of disease progression (HR 3,81; p = 0,010) after paclitaxel/cisplatin chemotherapy. CONCLUSIONS: The unfavorable genetic variants could facilitate carcinogenic process and once their carriers developed malignancy, their chances of survival were smaller. Our analyses also showed a strong gene-dosage effect with the decrease of progression-free survival for the carriers of two unfavorable genetic factors.

Associations between genes for killer immunoglobulin-like receptors and their ligands in patients with epithelial ovarian cancer.

Killer immunoglobulin-like receptors (KIRs) regulate function of NK cells and subsets of T cells. HLA class I molecules are ligands for inhibitory KIRs while specificity of activating KIRs is mainly unknown. Both KIR and HLA genotypes are highly polymorphic. In this study we analyzed associations of KIR and KIR ligand genes with the incidence and clinical course of epithelial ovarian cancer. DNA of 142 patients was analyzed for KIR genes and 103 samples were typed for HLA class I. Control group consisted of 200 healthy individuals, including 83 women, analyzed separately. The frequency of KIR genes in patients and controls were comparable. HLA-C group 1 (ligand for KIR2DL2/3) was more frequent in patients than in controls (86.4% vs. 67.5%, p=0.002). The frequency of KIR2DS4fl was higher in patients with endometrioid cancer (72.3%) compared with other histological subtypes (36.5%, p=0.004) and controls (29.5%, p=0.0001). KIR and KIR ligand genotype did not influence significantly the clinical course of the disease. We conclude that the genotype of KIR ligands is strongly associated with the incidence of epithelial ovarian cancer while KIR2DS4fl confers susceptibility to endometrioid subtype of the disease.

Genetic variation in ALCAM and other chromosomal instability genes in breast cancer survival.

Chromosomal instability is a hallmark of many cancers and it has a potential to predict clinical outcome of a cancer patient. We hypothesized that genes whose expression status differs between chromosomal stable and unstable breast tumors represent target genes for the identification of genetic variants predicting breast cancer (BC) risk, disease progression, and survival. We used a published list of 38 genes associated with chromosomal instability as a basis for searching potentially functional and informative tagging single nucleotide polymorphisms (SNPs). As a result, 33 SNPs in 16 genes were genotyped in a population-based series of 783 Swedish BC cases. Two SNPs in the ALCAM gene associated with BC-specific survival. For rs1044243, the HR was 4.35 (95% CI 1.34-14.18), and for rs1157, the HR was 3.42 (95% CI 1.32-8.83) for the homozygous carriers of the minor alleles. For the minor allele carriers of CCL18 SNP rs14304, we observed a significant association with aggressive tumor characteristics: large tumor size (OR 1.53, 95% CI 1.10-2.14), positive lymph node metastasis (OR 1.75, 95% CI 1.02-3.00), and high stage (OR 1.37, 95% CI 1.02-1.85). In a Polish population consisting of 506 familial/early onset BC cases, no association with event-free survival for the ALCAM SNPs nor any association with tumor characteristics for the CCL18 SNP were observed, suggesting either a chance finding in the Swedish population or population-based or etiological differences between sporadic and familial/early onset BC.

Single nucleotide polymorphisms in the 20q13 amplicon genes in relation to breast cancer risk and clinical outcome.

The 20q13 region is frequently amplified/overexpressed in breast tumours. However, the nature of this amplification/overexpression is unknown. Here, we investigated genetic variation in five 20q13 amplicon genes (MYBL2, AURKA, ZNF217, STK4 and PTPN1) and its impact on breast cancer (BC) susceptibility and clinical outcome. As a novel finding, four polymorphisms in STK4 (rs6017452, rs7271519) and AURKA (rs2273535, rs8173) associated with steroid hormone receptor status both in a Swedish population-based cohort of 783 BC cases and in a Polish familial/early onset cohort of 506 BC cases. In the joint analysis, the minor allele carriers of rs6017452 had more often hormone receptor positive tumours (OR 0.57, 95% CI 0.40-0.81), while homozygotes for the minor allele of rs7271519, rs2273535 and rs8173 had more often hormone receptor negative tumours (2.26, 1.30-3.39; 2.39, 1.14-5.01; 2.39, 1.19-4.80, respectively) than homozygotes for the common allele. BC-specific survival analysis of AURKA suggested that the Swedish carriers of the minor allele of rs16979877, rs2273535 and rs8173 might have a worse survival compared with the major homozygotes. The survival probabilities associated with the AURKA genotypes depended on the tumour phenotype. In the Swedish case-control study, associations with BC susceptibility were observed in a dominant model for three MYBL2 promoter polymorphisms (rs619289, P = 0.02; rs826943, P = 0.03 and rs826944, P = 0.02), two AURKA promoter polymorphisms (rs6064389, P = 0.04 and rs16979877, P = 0.02) and one 3'UTR polymorphism in ZNF217 (rs1056948, P = 0.01). In conclusion, our data confirmed the impact of the previously identified susceptibility locus and provided preliminary evidence for novel susceptibility variants in BC. We provided evidence for the first time that genetic variants at 20q13 may affect hormone receptor status in breast tumours and influence tumour aggressiveness and survival of the patients. Future studies are needed to confirm the prognostic value of our findings in the clinic.

Genetic variation in genes encoding for polymerase ζ subunits associates with breast cancer risk, tumour characteristics and survival.

Chromosomal instability is a known hallmark of many cancers. DNA polymerases represent a group of enzymes that are involved in the mechanism of chromosomal instability as they have a central function in DNA metabolism. We hypothesized that genetic variation in the polymerase genes may affect gene expression or protein configuration and by that cancer risk and clinical outcome. We selected four genes encoding for the catalytic subunits of the polymerases ß, δ, θ and ζ (POLB, POLD1, POLQ and REV3L, respectively) and two associated proteins (MAD2L2 and REV1) because of their previously reported association with chromosomal instability and/or tumorigenesis. We selected potentially functional and most informative tagging single nucleotide polymorphisms (SNPs) for genotyping in a population-based series of 783 Swedish breast cancer (BC) cases and 1562 controls. SNPs that showed a significant association in the Swedish population were additionally genotyped in a Polish population consisting of 506 familial/early onset BC cases and 568 controls. SNPs in all three polymerase ζ subunit genes associated either with BC risk or prognosis. Two SNPs in REV3L and one SNP in MAD2L2 associated with BC risk: rs462779 (multiplicative model: OR 0.79, 95% CI 0.68-0.92), rs3204953 (dominant model: OR 1.28, 95% CI 1.05-1.56) and rs2233004 (recessive model: OR 0.49, 95% CI 0.28-0.86). Homozygous carriers of the minor allele C of the third SNP in REV3L, rs11153292, had significantly worse survival compared to the TT genotype carriers (HR 2.93, 95% CI 1.34-6.44). Minor allele carriers of two REV1 SNPs (rs6761391 and rs3792142) had significantly more often large tumours and tumours with high histological grade and stage. No association was observed for SNPs in POLB, POLQ and POLD1. Altogether, our data suggest a significant role of genetic variation in the polymerase ζ subunit genes regarding the development and progression of BC.

Prevalence of the most frequent BRCA1 mutations in Polish population.

The purpose of our study was to establish the frequency and distribution of the four most common BRCA1 mutations in Polish general population and in a series of breast cancer patients. Analysis of the population frequency of 5382insC (c.5266dupC), 300T >G (p.181T >G), 185delAG (c.68_69delAG) and 3819del5 (c.3700_3704del5) mutations of the BRCA1 gene were performed on a group of respectively 16,849, 13,462, 12,485 and 3923 anonymous samples collected at birth in seven Polish provinces. The patient group consisted of 1845 consecutive female breast cancer cases. The most frequent BRCA1 mutation in the general population was 5382insC found in 29 out of 16,849 samples (0.17%). 300T >G and 3819del5 mutations were found in respectively 11 of 13,462 (0.08%) and four of 3923 (0.1%) samples. The population prevalence for combined Polish founder 5382insC and 300T >G mutations was 0.25% (1/400). The frequencies of 5382insC and 300T >G carriers among consecutive breast cancer cases were, respectively, 1.9% (35/1845) and 1.2% (18/1486). Comparing these data with the population frequency, we calculated the relative risk of breast cancer for 5382insC mutation at OR = 17 and for 300T >G mutation at OR = 26. Our results, based on large population studies, show high frequencies of founder 5382insC and 300T >G BRCA1 mutations in Polish general population. Carriage of one of these mutations is connected with a very high relative risk of breast cancer.

High-throughput genotyping of a common deletion polymorphism disrupting the TRY6 gene and its association with breast cancer risk.

BACKGROUND: Copy number polymorphisms caused by genomic rearrangements like deletions, make a significant contribution to the genomic differences between two individuals and may add to disease predisposition. Therefore, genotyping of such deletion polymorphisms in case-control studies could give important insights into risk associations. RESULTS: We mapped the breakpoints and developed a fluorescent fragment analysis for a deletion disrupting the TRY6 gene to exemplify a quick and cheap genotyping approach for such structural variants. We showed that the deletion is larger than predicted and encompasses also the pseudogene TRY5. We performed a case-control study to test an association of the TRY6 deletion polymorphism with breast cancer using a single nucleotide polymorphism which is in 100% linkage disequilibrium with the deletion. We did not observe an effect of the deletion on breast cancer risk (OR 1.05, 95% CI 0.71-1.56). CONCLUSION: Although we did not observe an association between the TRY6 deletion polymorphism and breast cancer risk, the identification and investigation of further deletions using the present approach may help to elucidate their effect on disease susceptibility.