Molecular prognostic and predictive factors of breast cancer

Despite advances in the early detection and treatment of breast cancer, it remains a challenge to identify which patients may experience a poor prognosis or respond poorly to treatment. Familial predisposition is a major risk factor of breast cancer and, perhaps, a modifier of patients’ survival. There is also evidence suggesting that hereditary factors may impact a patient’s response to treatment. However, the magnitude of the effect, and the molecular mechanism behind it, is largely unknown. In Finland, over 4,000 women on average are annually diagnosed with breast cancer and the majority of them undergo chemotherapy; they may or may not respond to the treatment. It is challenging to identify germline markers and tumor molecular profiles, which objectively predict prognosis and treatment response, and translate this information into cancer therapy. The aim of this thesis was to identify prognostic and predictive markers in breast cancer by investigating cancer-related networks as well as candidate genes of regulatory networks in invasive breast cancer cases. Taking both a network analysis approach as well as a candidate gene study, clinicopathological and survival association analyses were performed to (I) study the association of germline variations in TP53 network genes with breast cancer patients’ survival and treatment outcome; (II) investigate the impact of two-SNP interaction of NF-κB signaling network on predicting patients’ survival; (III) evaluate the association of NQO1 protein expression and NF-κB activation with clinicopathological features of the tumors, patients’ survival, and treatment outcome; and (IV) study the role of the miR-30 family in breast cancer patients’ survival and drug response. The germline variations were studied in collaboration with the Breast Cancer Association Consortium (BCAC). In Study I, the variations were initially analyzed in a set of DNA samples from 925 invasive breast cancer cases from Helsinki Breast Cancer Study (HEBCS) included in BCAC, and were further analyzed in pooled data of 4,701 cases from four independent studies (including HEBCS) contributing to BCAC. In Study II, the germline variations were studied in extensive pooled data of 30,431 cases from 24 independent studies participating in BCAC. In Studies III and IV, the tumor samples for immunohistochemical and miRNA in situ hybridization of 1,240 cases were from two series of 884 unselected Finnish invasive breast cancer patients and an additional 542 familial cases. Gene expression analysis was performed using microarray data of total RNA from 187 fresh frozen primary breast cancer tumors. Drug sensitivity screening tested the influence of miR-30 family members on the response of human breast cancer cell lines to two drugs, doxorubicin and lapatinib. In Study I, a significant interaction effect was found between germline variations in TP53-related genes, PRKAG2 (rs4726050) and MDM2 SNP309, with PRKAG2 (rs4726050) rare G allele showing a dose-dependent impact for superior breast cancer survival only among the MDM2 SNP309 rare G allele carriers. Also, PPP2R2B (rs10477313) rare A allele predicted increased survival after hormonal therapy. Further studies are warranted to clarify the impact of PRKAG2 and PPP2R2B on patients’ survival. In Study II, the SNP-SNP interaction test in the NF-κB activating pathway found two interacting SNP pairs, rs5996080-rs7973914 and rs17243893-rs57890595, which was associated with patients’ survival under recessive and dominant models of inheritance, respectively. While rs5996080 and rs7973914 were included in the study for representing the haplotype block harboring NF-κB activating genes, BAFFR and TNFR1/3, they physically reside in SREBF2 and SCNN1A, thus, the interacting effect found between these two loci may represent either of the genes. The dominant SNP pair, rs17243893 and rs57890595, represented TRAF2 and TRAIL-R4. Based on the published function of the interacting genes, and the in silico analysis of this study, the survival association of the identified SNP pairs may be a result of interplay between these gene pairs and their downstream influence on the dynamic of canonical and non-canonical NF-κB pathways. In Study III, the immunohistochemical staining analysis of NQO1 expression and NF-κB nuclear localization (inferred activity) did not find significant association between either of the proteins and patients’ survival or treatment outcome. However, an inverse correlation between NQO1 expression and NF-κB activity was observed in breast cancer tumors. The NQO1/NF-κB inverse correlation was also reflected in their association with ER status, as well as their correlation with gene expression. In Study IV, a significant association was found between the high expression of miR-30d and longer metastasis-free survival, particularly in subgroups of patients with high proliferative tumors, ER negativity, HER2 positivity, and among those who received chemotherapy. However, the high expression of miR-30 appeared to also correlate with the characteristics of aggressive tumors, i.e. higher grade, positive nodal status, and high proliferation (estimated by high Ki67). In a drug sensitivity screening test of all miR-30 family members, miR-30a–e sensitized the human breast cancer cell lines to doxorubicin. Also, in the HER2-positive HCC1954 cell line, miR-30d sensitized the cells to lapatinib. The pathway enrichment analysis of miR-30 family members in the METABRIC gene expression dataset revealed that high levels of miR-30 family occurred simultaneously with low expressions of genes involved in cell movements, consistent with the observed association with longer metastasis-free survival. The result of this work suggests prognostic/predictive potentials for candidate genes in cancer-related networks (TP53 and NF-κB), as well as regulatory networks (microRNAs), which warrant further investigations.Suomessa rintasyöpä diagnosoidaan keskimäärin yli 4000 naisella vuosittain ja suurin osa heistä saa kemoterapiaa; he saattavat reagoida hoitoon tai eivät. On haastavaa löytää geneettinen merkki, joka ennustaisi ennusteen sekä hoitovasteen. Yhtä haastavaa on soveltaa näitä tietoja syöpähoidossa. Tämän väitöskirjan tarkoituksena oli löytää uusia rintasyövän geneettisiä markkereita tutkimalla syöpään liittyviä geenejä kliinisellä, patologisella ja potilaiden eloonjääntianalyysillä. Tässä opinnäytetyössä käytetty aineisto sisälsi 1240 kasvainnäytettä Suomesta ja 30 431 DNA-näytettä Breast Cancer Association Consortiumilta (BCAC). Merkittävä vuorovaikutus havaittiin TP53: een liittyvien geenien, PRKAG2:n (rs4726050) ja MDM2 SNP309, mikä liittyi potilaan parempaan eloonjäämiseen. PPP2R2B (rs10477313) ennusti myös pidemmän eloonjäämisen hormonaalisen hoidon jälkeen. Lisätutkimuksia tarvitaan tutkimaan PRKAG2:n ja PPP2R2B:n vaikutusta potilaan eloonjäämiseen. NFkB-pathway tutkimuksessa SNP-SNP-vuorovaikutustesti löysi kaksi SNP-paria, rs5996080-rs7973914 ja rs17243893-rs57890595, jotka liittyivät potilaiden eloonjäämiseen. Nämä SNP: t osoittavat BAFFR: n, TNFR1 / 3: n, SREBF2: n, SCNN1A: n, TRAF2: n tai TRAIL-R4: n mahdollisen yhteyden potilaan eloonjäämiseen. Tunnistettujen SNP-parien selviytymisyhteys voi johtua näiden geeniparien välisestä vuorovaikutuksesta ja niiden alavirran vaikutuksesta kanonisten ja ei-kanonisten NF-KB-reittien dynamiikkaan. NQO1- ja NF-KB-ilmentymisen immunohistokemiallinen analyysi ei löytänyt merkittävää yhteyttä näiden proteiinien ilmentymisen ja potilaan eloonjäämisen tai hoidon lopputuloksen välillä. Rintasyöpäkasvaimissa havaittiin kuitenkin käänteinen korrelaatio NQO1-ilmentymisen ja NF-KB-aktiivisuuden välillä. miR-30d:n ilmeneminen assosioitui potilaiden parempaan eloonjäämiseen, erityisesti sellaisten potilaiden alaryhmissä, joilla on korkea Ki67, ER-negatiivisuus, HER2-positiivisuus, ja kemoterapiaa saaneiden potilaiden joukossa. MiR-30a-e herkistää myös ihmisen rintasyövän solulinjat doksorubisiinille ja lapatinibille. Tämän työn tulos viittaa ennustepotentiaaliin geeneihin syöpään liittyvissä verkoissa (TP53 ja NF-KB) sekä säätelyverkoissa (mikroRNA: t). Näiden tulosten vahvistamiseksi tarvitaan lisää tutkimuksia

MiR-34a Expression Has an Effect for Lower Risk of Metastasis and Associates with Expression Patterns Predicting Clinical Outcome in Breast Cancer

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NQO1 expression correlates inversely with NFκB activation in human breast cancer

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High miR-30 Expression Associates with Improved Breast Cancer Patient Survival and Treatment Outcome

Deregulated miRNA expression has been suggested in several stages of breast cancer pathogenesis. We have studied the miR-30 family, in particular miR-30d, in relation to breast cancer patient survival and treatment outcomes. With tumor specimens from 1238 breast cancer patients, we analyzed the association of miR-30d expression with tumor characteristics with the 5-year occurrence of breast cancer-specific death or distant metastasis (BDDM), and with 10-year breast cancer survival (BCS). We conducted a two-stage drug-screen to investigate the impact of miR-30 family members (miR-30a-30e) on sensitivity to doxorubicin and lapatinib in six breast cancer cell lines HCC1937, HCC1954, MDA-MB-361, MCF7, MDA-MB-436 and CAL-120, using drug sensitivity scores (DSS) to compare the miR-30 family mimics to their specific inhibitors. The study was complemented with Ingenuity Pathway Analysis (IPA) with the METABRIC data. We found that while high miR-30d expression is typical for aggressive tumors, it predicts better metastasis-free (pBDDM = 0.035, HR = 0.63, 95% CI = 0.4–0.9) and breast cancer-specific survival (pBCS = 0.018, HR = 0.61, 95% CI = 0.4–0.9), especially in HER2-positive (pBDDM = 0.0009), ER-negative (pBDDM = 0.003), p53-positive (pBDDM = 0.011), and highly proliferating (pBDDM = 0.0004) subgroups, and after adjuvant chemotherapy (pBDDM = 0.035). MiR-30d predicted survival independently of standard prognostic markers (pBDDM = 0.0004). In the drug-screening test, the miR-30 family sensitized the HER2-positive HCC1954 cell line to lapatinib (p < 10−2) and HCC1937, MDA-MB-361, MDA-MB-436 and CAL120 to doxorubicin (p < 10−4) with an opposite impact on MCF7. According to the pathway analysis, the miR-30 family has a suppressive effect on cell motility and metastasis in breast cancer. Our results suggest prognostic and predictive potential for the miR-30 family, which warrants further investigation

High miR-30 Expression Associates with Improved Breast Cancer Patient Survival and Treatment Outcome

Deregulated miRNA expression has been suggested in several stages of breast cancer pathogenesis. We have studied the miR-30 family, in particular miR-30d, in relation to breast cancer patient survival and treatment outcomes. With tumor specimens from 1238 breast cancer patients, we analyzed the association of miR-30d expression with tumor characteristics with the 5-year occurrence of breast cancer-specific death or distant metastasis (BDDM), and with 10-year breast cancer survival (BCS). We conducted a two-stage drug-screen to investigate the impact of miR-30 family members (miR-30a-30e) on sensitivity to doxorubicin and lapatinib in six breast cancer cell lines HCC1937, HCC1954, MDA-MB-361, MCF7, MDA-MB-436 and CAL-120, using drug sensitivity scores (DSS) to compare the miR-30 family mimics to their specific inhibitors. The study was complemented with Ingenuity Pathway Analysis (IPA) with the METABRIC data. We found that while high miR-30d expression is typical for aggressive tumors, it predicts better metastasis-free (pBDDM = 0.035, HR = 0.63, 95% CI = 0.4–0.9) and breast cancer-specific survival (pBCS = 0.018, HR = 0.61, 95% CI = 0.4–0.9), especially in HER2-positive (pBDDM = 0.0009), ER-negative (pBDDM = 0.003), p53-positive (pBDDM = 0.011), and highly proliferating (pBDDM = 0.0004) subgroups, and after adjuvant chemotherapy (pBDDM = 0.035). MiR-30d predicted survival independently of standard prognostic markers (pBDDM = 0.0004). In the drug-screening test, the miR-30 family sensitized the HER2-positive HCC1954 cell line to lapatinib (p < 10−2) and HCC1937, MDA-MB-361, MDA-MB-436 and CAL120 to doxorubicin (p < 10−4) with an opposite impact on MCF7. According to the pathway analysis, the miR-30 family has a suppressive effect on cell motility and metastasis in breast cancer. Our results suggest prognostic and predictive potential for the miR-30 family, which warrants further investigation

High miR-30 Expression Associates with Improved Breast Cancer Patient Survival and Treatment Outcome

Simple Summary Previous research on the miR-30 family and breast cancer patient survival and on miR-30-related chemosensitivity prompted us to design a comprehensive study on the role of the miR-30 family in general and on miR-30d in particular in breast cancer. We present a study consisting of a tumor microarray analysis of 1238 breast cancer patients, a survival analysis, a drug-sensitivity screen with six breast cancer cell lines, and an in-silico pathway analysis. In our analysis, high miR-30d expression was associated with improved survival in breast cancer patients with aggressive tumor phenotypes. In the drug-sensitivity analysis, ectopic expression of miR-30 family members sensitized the cell lines to the treatment. The pathway analysis based on miRNA and mRNA expression in the METABRIC data suggested that the miR-30 family may have an inhibitory role in pathways contributing to EMT and metastasis. Our results suggest prognostic and predictive potential for the miR-30 family for further investigation. Deregulated miRNA expression has been suggested in several stages of breast cancer pathogenesis. We have studied the miR-30 family, in particular miR-30d, in relation to breast cancer patient survival and treatment outcomes. With tumor specimens from 1238 breast cancer patients, we analyzed the association of miR-30d expression with tumor characteristics with the 5-year occurrence of breast cancer-specific death or distant metastasis (BDDM), and with 10-year breast cancer survival (BCS). We conducted a two-stage drug-screen to investigate the impact of miR-30 family members (miR-30a-30e) on sensitivity to doxorubicin and lapatinib in six breast cancer cell lines HCC1937, HCC1954, MDA-MB-361, MCF7, MDA-MB-436 and CAL-120, using drug sensitivity scores (DSS) to compare the miR-30 family mimics to their specific inhibitors. The study was complemented with Ingenuity Pathway Analysis (IPA) with the METABRIC data. We found that while high miR-30d expression is typical for aggressive tumors, it predicts better metastasis-free (p(BDDM) = 0.035, HR = 0.63, 95% CI = 0.4-0.9) and breast cancer-specific survival (p(BCS) = 0.018, HR = 0.61, 95% CI = 0.4-0.9), especially in HER2-positive (p(BDDM) = 0.0009), ER-negative (p(BDDM) = 0.003), p53-positive (p(BDDM) = 0.011), and highly proliferating (p(BDDM) = 0.0004) subgroups, and after adjuvant chemotherapy (p(BDDM) = 0.035). MiR-30d predicted survival independently of standard prognostic markers (p(BDDM) = 0.0004). In the drug-screening test, the miR-30 family sensitized the HER2-positive HCC1954 cell line to lapatinib (p < 10(-2)) and HCC1937, MDA-MB-361, MDA-MB-436 and CAL120 to doxorubicin (p < 10(-4)) with an opposite impact on MCF7. According to the pathway analysis, the miR-30 family has a suppressive effect on cell motility and metastasis in breast cancer. Our results suggest prognostic and predictive potential for the miR-30 family, which warrants further investigation

FANCM c.5101C > T mutation associates with breast cancer survival and treatment outcome

Breast cancer (BC) is a heterogeneous disease, and different tumor characteristics and genetic variation may affect the clinical outcome. The FANCM c.5101C> T nonsense mutation in the Finnish population associates with increased risk of breast cancer, especially for triple-negative breast cancer patients. To investigate the association of the mutation with disease prognosis, we studied tumor phenotype, treatment outcome, and patient survival in 3,933 invasive breast cancer patients, including 101 FANCM c.5101C> T mutation carriers and 3,832 non-carriers. We also examined association of the mutation with nuclear immunohistochemical staining of DNA repair markers in 1,240 breast tumors. The FANCM c.5101C>T mutation associated with poor 10-year breast cancer-specific survival (hazard ratio (HR) 51.66, 95% confidence interval (CI) 1.09-2.52, p=0.018), with a more pronounced survival effect among familial cases (HR=2.93, 95% CI 1.5-5.76, p=1.80 x 10 23). Poor disease outcome of the carriers was also found among the estrogen receptor (ER) positive subgroup of patients (HR=1.8, 95% CI 1.09-2.98, p=0.021). Reduced survival was seen especially among patients who had not received radiotherapy (HR=3.43, 95% CI 1.6-7.34, p=1.50x10(-3)) but not among radiotherapy treated patients (HR=1.35, 95% CI 0.82-2.23, p=0.237). Significant interaction was found between the mutation and radiotherapy (p=0.040). Immunohistochemical analyses show that c.5101C> T carriers have reduced PAR-activity. Our results suggest that FANCM c.5101C>T nonsense mutation carriers have a reduced breast cancer survival but postoperative radiotherapy may diminish this survival disadvantage.Peer reviewe

SNP-SNP interaction analysis of NF-kappa B signaling pathway on breast cancer survival

In breast cancer, constitutive activation of NF-kappa B has been reported, however, the impact of genetic variation of the pathway on patient prognosis has been little studied. Furthermore, a combination of genetic variants, rather than single polymorphisms, may affect disease prognosis. Here, in an extensive dataset (n = 30,431) from the Breast Cancer Association Consortium, we investigated the association of 917 SNPs in 75 genes in the NF-kappa B pathway with breast cancer prognosis. We explored SNP-SNP interactions on survival using the likelihood-ratio test comparing multivariate Cox' regression models of SNP pairs without and with an interaction term. We found two interacting pairs associating with prognosis: patients simultaneously homozygous for the rare alleles of rs5996080 and rs7973914 had worse survival (HRinteraction 6.98, 95% CI= 3.3-14.4, P=1.42E-07), and patients carrying at least one rare allele for rs17243893 and rs57890595 had better survival (HRinteraction 0.51, 95% CI= 0.3-0.6, P = 2.19E-05). Based on in silico functional analyses and literature, we speculate that the rs5996080 and rs7973914 loci may affect the BAFFR and TNFR1/TNFR3 receptors and breast cancer survival, possibly by disturbing both the canonical and non-canonical NF-kappa B pathways or their dynamics, whereas, rs17243893-rs57890595 interaction on survival may be mediated through TRAF2-TRAIL-R4 interplay. These results warrant further validation and functional analyses.Peer reviewe