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