Factors and Mechanisms in Hereditary Breast Cancer Predisposition

Project Leader
Prof. Robert Winqvist, Ph.D.
Laboratory of Cancer Genetics and Tumour Biology, Cancer and Translational Medicine Research Unit, Biocenter Oulu and Faculty of Medicine, University of Oulu

Background and Significance

Breast cancer is a heterogeneous disease and is the most common malignancy among women. At present, about one in eight women will eventually develop breast cancer and despite significant advances in detection and treatment, breast cancer still remains a major cause of death in the Western world.

Accumulation of various genomic lesions plays a major role both in the initial development and in the progression of breast cancer. Increased genome instability is recognized as a hallmark of cancer. Although most cases of female breast cancer appear to be “sporadic”, between 5 and 10% of the cases are estimated to have a strong hereditary background, due to the transmission of certain harmful genetic risk factors, which make it more plausible for a mutation carrier to develop a malignancy – often at a much earlier age than in the general population. Unfortunately, however, the known major susceptibility genes BRCA1 and BRCA2 explain only about 20% of the familial breast cancer cases, and altogether the known high-penetrance heritable risk factors contribute to approximately 25% of these cases, leaving the remaining 75% unexplained. Therefore, the remaining high-risk genetic factors also need to be identified. In addition to high-penetrance susceptibility factors, more recently multiple and more commonly occurring moderate- as well as low-penetrance constitutional cancer-predisposing variants have been identified. Together with various environmental and life-style risk factors, all of these kinds of constitutional disease-predisposing factors are likely to play very significant roles in the aetiology of breast cancer.

Shared features of many of the presently known cancer susceptibility genes include the nature of their protein products, which typically perform important functions in cell-cycle control, DNA double-strand damage response pathways, as well as in other genome integrity control utilities. Therefore, it is plausible that other genes influencing these essential cellular pathways would also be targets as regards germline mutations associated with an increased cancer risk.

The increased knowledge of the biology of tumour formation and cancer progression can subsequently be utilized to develop more effective tools for diagnostic, prognostic and therapeutic purposes. Effective identification of individuals at significantly increased hereditary disease risk, together with intensified surveillance of such individuals for early-stage malignancy detection is imperative for successful treatment and favourable prognosis.

Recent Progress

The general aim of our research is to identify the most important genomic factors involved in the development and also in the progression of breast cancer. Additionally, a better understanding of the biological consequences of the various aberrations observed in these genes will be helpful for creating more effective means of disease prevention, diagnosis and treatment. Our investigations focus particularly on hereditary predisposition to breast cancer and on unravelling novel susceptibility factors, mainly using a case-control study design, supplemented by the utilisation of geographically matched incident hospital-based breast cancer cases, unselected for a family history of cancer.

By studying Northern Finnish breast cancer families that had previously tested negative for heterozygous germline mutations in the BRCA1 and BRCA2 genes, during the past few years we have been able to uncover several important and novel cancer susceptibility genes (e.g. PALB2, RAD50, NBS1, RAP80, ABRAXAS, MCPH1). Furthermore, in studies that have also included the ATM gene, we have been able to provide evidence of involvement of both haploinsufficiency and dominant-negative allele behaviour in heritable cancer risk. We have also carried out some initial investigations to characterise the molecular, physiological and clinical consequences of the observed genetic aberrations, those of PALB2 in particular, and more detailed assessments are currently in progress.

We reported in Nature in 2007 the discovery of PALB2, a novel breast cancer susceptibility gene, the protein product of which regulates key features of the physiological responses to DNA damage mediated by the BRCA2 susceptibility product. In addition, PALB2 directly interacts with BRCA1, the other major breast cancer susceptibility gene product. In fact, PALB2 bridges the interplay between these two biologically essential proteins.

In a subsequent study published in Clinical Cancer Research in 2008, we observed that heterozygous carriers of this relatively common Finnish PALB2 founder mutation on average displayed a breast cancer risk of 40% by age 70, which is a substantially higher risk estimate than had been originally anticipated. The number is similar to that determined for BRCA2 (45%), but somewhat lower than that for BRCA1 (76%). Consequently, PALB2 germline mutations are likely to have important physiological as well as clinical implications. Interestingly, there is now evidence for the existence of many different breast cancer predisposition-related germline mutations in PALB2 in various populations worldwide (e.g. Finland, the UK, the Netherlands, Belgium, Italy, Germany, Poland, Canada, the USA, South Africa, China and Australia), underlining the significant contribution of PALB2 dysfunction in hereditary-based breast cancer development.

Testing for mutations in the BRCA1 and BRCA2 genes among high-risk breast cancer patients has become routine practice in clinical genetics. Unfortunately, however, at present the genetic background of the majority of cases coming to the clinics remains currently unexplained, making genetic counselling rather challenging. We have carried out a study evaluating the need for routine clinical testing for the relatively common PALB2 c.1592delT founder mutation in BRCA-negative Northern Finnish breast cancer families (BMC Med Genet 2013). The study revealed multiple PALB2 c.1592delT mutation carriers among the studied high-risk breast cancer cases. Subsequently, all of our exciting initial findings regarding PALB2 have been further confirmed in a large comprehensive international meta-analysis of families with various heterozygous loss-of-function PALB2 mutations, coordinated by the PALB2 Interest Group (NEJM 2014). The observed breast cancer risk of female PALB2 mutation carriers varied between 33 and 58%, and the greatest risk was seen for persons with close relatives with breast cancer. All of the currently available information supports the notion that besides BRCA1 and BRCA2, PALB2 is also a high-risk susceptibility gene for female breast cancer, thus warranting its inclusion in the standard genetic counselling mutation screening protocol offered to individuals at an increased risk of hereditary breast cancer.

In a recent study (PLoS Genetics 2016), we have discovered yet another novel gene, MCPH1, involved in hereditary breast cancer susceptibility. As many as 21 cancer families from Northern Finland have a founder germline mutation in this gene identified through massive parallel sequencing of hundreds of DNA damage response genes. Owing to significantly elevated genomic instability, heterozygous mutation carriers exhibited a 3- to 8-fold increased risk of cancer. Besides breast cancer, one third of the families with MCPH1 mutation also exhibited brain tumours and/or sarcomas. Next it will be important to explore the role of MCPH1 mutations in relation to cancer susceptibility in other populations.  

Although females carrying a heterozygous germline mutation in BRCA1, BRCA2, PALB2, MCPH1 or some other key gene are associated with an increased propensity to develop breast cancer, the mechanistic details of how this adverse effect is brought about have remained largely obscure. For instance, it has been previously shown that the BRCA1, BRCA2 and PALB2 proteins together regulate cellular repair of DNA breaks by homologous recombination. In order to investigate the molecular basis and biological mechanisms that underlie hereditable cancer risk we first focused on the PALB2 gene, using lymphoblastoid cell lines derived from Finnish familial breast cancer patients that all harbour the same heterozygous c.1592delT truncation mutation. This mutation occurs in as much as 1% of all Finnish breast cancer patients and is expected to behave similarly to other PALB2 truncation mutations worldwide that are associated with breast cancer predisposition.

The mutation carrier cells still have one functional copy of the PALB2 gene and contained approximately half the amount of PALB2 protein compared with that in healthy control cells. The carrier cells showed accelerated duplication of their DNA. We discovered that PALB2 mutation carrier cells started replication twice as often as unaffected control cells, in this way ‘rushing’ through the process, utilizing part of the normally dormant origins in the absence of an external disturbance. This leads to problems, since DNA replication seems to stall more regularly in the mutation carrier cells. What is more, these cells are no longer able to react upon disturbances, since part of the back-up of replication start sites is already exhausted during normal growth. Furthermore, growing cells use multiple consecutive checkpoints to survey the genome for damage and to ensure that one stage of the cell division cycle has been completed successfully before the next stage is initiated. In cases of DNA damage, PALB2 mutation carrier cells first show a robust checkpoint response, but in contrast to healthy control cells, the carrier cells fail to maintain this checkpoint response and resume cell growth despite persisting damage. This leads to genomic instability that is the driving force behind the early stages of cancer development. The fact that metaphases from short-term cultivation of primary blood lymphocytes of heterozygous PALB2 mutation carriers show an increased number of chromosome aberrations demonstrates that the genome destabilisation observed in our experiments is also operational at the organism level. As PALB2 haploinsufficiency has already been found to cause aberrant DNA replication/damage response resulting in increased genomic instability, it would suggest that functional loss of the remaining wild-type allele, as defined by the Knudson two-hit hypothesis, may not always be a prerequisite for malignancy development. Altogether, these findings provide a new and exciting mechanism for the early stages of breast cancer development that may also apply to mutations in other genes indicated in hereditary cancer predisposition (Nat Commun 2013, Oncogene 2015).

We have also successfully carried out mouse modelling studies in connection with constitutional PALB2 gene defects (Oncogene 2010). Mice lacking proper Palb2 function die as embryos and display defective mesoderm differentiation, suggesting an essential role for Palb2 in embryogenesis, possibly in concert with extracellular matrix influences. Mice lacking Palb2 display failure in epithelial-mesenchymal transition (EMT), a feature shared with many cancers. Follow-up investigations concerning these important findings are on their way.

For several years we have actively participated in various collaborative international studies via BCAC, TNBBC and PALB2 Interest Group consortium initiatives. Many of these investigations have been very successful, especially in revealing major contributions of various novel low-penetrance susceptibility alleles in the aetiology of breast cancer, non-familial disease in particular. Importantly, the same massive case-control studies have also uncovered the existence of a new class of constitutional genetic variants, namely those that are helpful in counteracting the development of cancer. Furthermore, these consortium studies have revealed clinically important associations between patient genotype and disease phenotype and outcome.

Future Goals

The main aims of our ongoing studies include the following:

1. We will try to identify additional constitutional breast cancer susceptibility alleles by multiple and partly parallel approaches: screening of candidate genes for high- or moderate-penetrance mutations, and genome-wide SNP association studies to identify relatively frequently occurring low-penetrance cancer risk alleles or risk haplotypes, including polyallelic clusters (mainly through international consortium collaboration). Furthermore, genome-wide exome sequencing and targeted high-throughput deep-sequencing will be utilised in order to obtain a more comprehensive view of the involved germline defects in predisposition to breast cancer.

2. We will continue to use lymphoblastoid and fibroblast cell lines derived from various germline mutation-positive individuals to characterise the molecular and physiological effects of these cancer-associated genetic defects in yet greater detail. Case and control cell lines will be challenged with various doses of γ-radiation, etoposide or hydroxyurea, for instance, and compared as regards molecular responses and DNA damage correction capacity and accuracy. In addition, in vitro 2D and 3D epithelial breast cancer modelling as well as disease modelling in mice will be used.

3. Various comprehensive next-generation sequencing approaches both at the DNA and RNA level will be used in order to define the high-resolution clonal cell population-specific aberration profiles of tumour specimens and other suitable tissue specimens derived from individuals displaying various constitutional gene aberrations originally identified in our laboratory in Oulu. In addition, CRISPR/Cas9 genome editing will be used to replicate naturally occurring key breast cancer susceptibility gene defects as well as somatic mutations and to explore their molecular and biological consequences under carefully monitored experimental conditions.

Ultimately, we hope to unravel as many novel hereditary risk factors as we can and assess their possible combinatory effects on the development of breast cancer in particular. By characterising the molecular, physiological and clinical effects of these genetic and epigenetic lesions we aim to add to the knowledge of critical biological pathways and mechanisms related to malignancy development, thus contributing towards better means of assessing disease risk, and towards the prevention and improved diagnostics, monitoring and treatment of cancer.

Publications 2016-

Couch FJ, Kuchenbaecker KB, Michailidou K, Mendoza-Fandino GA, Nord S, et al (incl Winqvist R, Pylkäs K). Identification of four novel susceptibility loci for oestrogen receptor negative breast cancer. Nat Commun 7:11375, 2016.

Darabi H, Beesley J, Droit A, Kar S, Nord S, et al (incl Winqvist R, Pylkäs K). Fine scale mapping of the 17q22 breast cancer locus using dense SNPs, genotyped within the Collaborative Oncological Gene-Environment Study (COGS). Sci Rep 6:32512, 2016.

Dunning AM, Michailidou K, Kuchenbaecker KB, Thompson D, French JD, et al (incl Winqvist R, Pylkäs K). Breast cancer risk variants at 6q25 display different phenotype associations and regulate ESR1, RMND1 and CCDC170. Nat Genet 48:374-386, 2016.

Easton DF, Lesueur F, Decker B, Michailidou K, Li J, et al (incl Winqvist R, Pylkäs K). No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk: implications for gene panel testing. J Med Genet 53:298-309, 2016.

Ghoussaini M, French JD, Michailidou K, Nord S, Beesley J, et al (incl Winqvist R, Pylkäs K). Evidence that the 5p12 Variant rs10941679 Confers Susceptibility to Estrogen-Receptor-Positive Breast Cancer through FGF10 and MRPS30 Regulation. Am J Hum Genet 99:903-911, 2016.

Guo Y, Warren Andersen S, Shu XO, Michailidou K, Bolla MK, et al (incl Winqvist R, Pylkäs K). Genetically Predicted Body Mass Index and Breast Cancer Risk: Mendelian Randomization Analyses of Data from 145,000 Women of European Descent. PLoS Med 13:e1002105, 2016.

Hamdi Y, Soucy P, Adoue V, Michailidou K, Canisius S, et al (incl Winqvist R, Pylkäs K). Association of breast cancer risk with genetic variants showing differential allelic expression: Identification of a novel breast cancer susceptibility locus at 4q21. Oncotarget 6;7(49):80140-80163, 2016.

Horne HN, Chung CC, Zhang H, Yu K, Prokunina-Olsson L, Michailidou K, et al (incl Winqvist R, Jukkola-Vuorinen A). Fine-Mapping of the 1p11.2 Breast Cancer Susceptibility Locus. PLoS One 11(8):e0160316, 2016.

Kar SP, Beesley J, Amin Al Olama A, Michailidou K, Tyrer J, et al (incl Winqvist R). Genome-Wide Meta-Analyses of Breast, Ovarian, and Prostate Cancer Association Studies Identify Multiple New Susceptibility Loci Shared by at Least Two Cancer Types. Cancer Discov 6:1052-1067, 2016.

Kiiski JI, Fagerholm R, Tervasmäki A, Pelttari LM, Khan S, Jamshidi M, Mantere T, Pylkäs K, Bartek J, Bartkova J, Mannermaa A, Tengström M, Kosma VM, Winqvist R, Kallioniemi A, Aittomäki K, Blomqvist C, Nevanlinna H. FANCM c.5101C>T mutation associates with breast cancer survival and treatment outcome. Int J Cancer 139:2760-2770, 2016.

Lawrenson K, Kar S, McCue K, Kuchenbaeker K, Michailidou K, et al (incl Winqvist R, Pylkäs K). Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast-ovarian cancer susceptibility locus. Nat Commun 7:12675, 2016.

Lei J, Rudolph A, Moysich KB, Behrens S, Goode EL, et al (incl Winqvist R, Grip M). Genetic variation in the immunosuppression pathway genes and breast cancer susceptibility: a pooled analysis of 42,510 cases and 40,577 controls from the Breast Cancer Association Consortium. Hum Genet 135:137-154, 2016.

Liu J, Lončar I, Collée JM, Bolla MK, Dennis J, et al (incl Winqvist R, Pylkäs K). rs2735383, located at a microRNA binding site in the 3'UTR of NBS1, is not associated with breast cancer risk. Sci Rep 6:36874, 2016.

Mantere T, Winqvist R*, Kauppila S, Grip M, Jukkola-Vuorinen A, Tervasmäki A, Rapakko K, Pylkäs K* (shared senior authorship). Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility. PLoS Genet 12:e1005816, 2016.

Meeks HD, Song H, Michailidou K, Bolla MK, Dennis J, et al (incl Winqvist R, Pylkäs K). BRCA2 Polymorphic Stop Codon K3326X and the Risk of Breast, Prostate, and Ovarian Cancers. J Natl Cancer Inst 108:djv315, 2016.

Muranen T, Blomqvist C, Dörk T, Jakubowska A, Heikkilä P, et al (incl Winqvist R, Pylkäs K). Patient survival and tumor characteristics associated with CHEK2:p.I157T - findings from the Breast Cancer Association Consortium. Breast Cancer Res 18:98, 2016.

Obermeier K, Sachsenweger J, Friedl TW, Pospiech H, Winqvist R*, Wiesmüller L* (shared senior authorship). Heterozygous PALB2 c.1592delT mutation channels DNA double-strand break repair into error-prone pathways in breast cancer patients. Oncogene 35:3796-3806, 2016.

Pelttari LM, Khan S, Vuorela M, Kiiski JI, Vilske S, Nevanlinna V, Ranta S, Schleutker J, Winqvist R, et al (Vuorela M). RAD51B in Familial Breast Cancer. PLoS One 11:e0153788, 2016.

Petridis C, Brook MN, Shah V, Kohut K, Gorman P, et al (incl Winqvist R, Pylkäs K). Genetic predisposition to ductal carcinoma in situ of the breast. Breast Cancer Res 18:22, 2016.

Shi J, Zhang Y, Zheng W, Michailidou K, Ghoussaini M, Bolla MK, et al (incl Winqvist R). Fine-scale mapping of 8q24 locus identifies multiple independent risk variants for breast cancer. Int J Cancer 139:1303-1317, 2016.

Southey MC, Goldgar DE, Winqvist R, Pylkäs K, Couch F, et al. PALB2, CHEK2 and ATM rare variants and cancer risk: data from COGS. J Med Genet 53:800-811, 2016.

Wyszynski A, Hong CC, Lam K, Michailidou K, Lytle C, Yao S, et al (incl Winqvist R, Pylkäs K). An intergenic risk locus containing an enhancer deletion in 2q35 modulates breast cancer risk by deregulating IGFBP5 expression. Hum Mol Genet 25:3863-3876, 2016.

Zeng C, Guo X, Long J, Kuchenbaecker KB, Droit A, et al (incl Winqvist R). Identification of independent association signals and putative functional variants for breast cancer risk through fine-scale mapping of the 12p11 locus. Breast Cancer Res 18:64, 2016.

Zhao Z, Wen W, Michailidou K, Bolla MK, Wang Q, et al (incl Winqvist R, Pylkäs K). Association of genetic susceptibility variants for type 2 diabetes with breast cancer risk in women of European ancestry. Cancer Causes Control 27:679-693, 2016.

Milne RL, Kuchenbaecker KB, Michailidou K, Beesley J, Kar S, et al (incl. Winqvist R, Pylkäs K of OBCS, Oulu). Ten variants associated with risk of estrogen receptor negative breast cancer. Nat Genet, in press.

Research Group Members

Project Leader:
Robert Winqvist, Ph.D., Professor (University of Oulu)

Senior and Post-doctoral Investigators:
Katri Pylkäs, Ph.D. (Biocenter Oulu)
Hellevi Peltoketo, Ph.D. (Academy of Finland)
Maria Haanpää, M.D., Ph.D. (from October 2015, 2-year Sigrid Jusélius Post-doctoral Fellowship for research at Stanford University Medical School, USA)
Leila Eshraghi, Ph.D. (from June 1, Sigrid Jusélius Foundation)
Arja Jukkola-Vuorinen, M.D., Ph.D. (Oulu University Hospital)
Mervi Grip, M.D. (Oulu University Hospital)
Saila Kauppila, M.D., Ph.D. (Oulu University Hospital)
Jukka Moilanen, M.D., Ph.D. (Oulu University Hospital)

Ph.D. Students:
Muthiah Bose, M.Sc. (Academy of Finland)
Tuomo Mantere, M.Sc. (UniOGS, Biocenter Oulu Doctoral Programme)
Raman Devarajan, M.Sc. (jointly with Taina Pihlajaniemi group, Academy of Finland)
Anna Tervasmäki, M.Sc. (UniOGS, Biocenter Oulu Doctoral Programme)
Niina Laurila, M.Sc. (Finnish Cultural Foundation, Finnish Cancer Foundation)
Hanna Tuppurainen, M.Sc. (Finnish Cancer Foundation, Sigrid Jusélius Foundation)
Juliane Sachsenweger, M.Sc. (jointly with Lisa Wiesmüller, International Graduate School in Molecular Medicine Ulm, Germany, and Biocenter Oulu Doctoral Programme)

Laboratory Technicians, 2 (Academy of Finland and Biocenter Oulu)

Main source of salary in brackets.

Foreign Scientists, 4

National and International Activities

Centre of Excellence in Cell-Extracellular Matrix Research, Academy of Finland Programme for 2012–2017

Taina Pihlajaniemi, Director; Johanna Myllyharju, Vice director; other Group leaders: Seppo Vainio, Robert Winqvist, Lauri Eklund, Aki Manninen.

The International Graduate School in Molecular Medicine Ulm, Germany, and UniOGS/Biocenter Oulu Doctoral Programme, University of Oulu: Joint Ph.D. project supervision 10.2014-9.2017

Prof. Lisa Wiesmüller and Robert Winqvist: M.Sc. Juliane Sachsenweger’s Ph.D. project.

Group Members Who Spent More Than Two Weeks in Foreign Laboratories During 2016

Maria Haanpää, M.D., Ph.D. (from October 2015, 2-year Sigrid Jusélius Post-doctoral Fellowship, Prof. James Ford Group / Program for Clinical Cancer Genetics and Genomics, Stanford University Medical School, USA).

EU Projects

EU COGS collaborative initiative on hereditary susceptibility to cancer. The Oulu Breast Cancer Study (OBCS), represented by our team, is a member of the BCAC (breast cancer association consortium) sub-study of COGS that is coordinated by Prof. Douglas Easton (Cambridge, UK), performing microarray-based, genome-wide SNP studies to reveal novel disease association factors as well as patterns of occurrence and characteristic phenotypic and clinical features.

Other International Consortium Activities

The clinical parameter stratification approach also includes participation in the Triple-Negative Breast Cancer Consortium (TNBCC) coordinated by Prof. Fergus Couch (Rochester, USA). The studies of this consortium are focused on individuals with breast cancer negative for the oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, and will provide important insights into the aetiology of different breast tumour subtypes.

The “PALB2 Interest Group” was established in 2007 through the joint initiative of Prof. William Foulkes (coordinator; Montreal, Canada), Prof. Melissa Southey (Melbourne, Australia) and Prof. Robert Winqvist for carrying out and coordinating comprehensive world-wide PALB2-related research projects. These activities include further characterisation of the occurrence and role of PALB2 defects in hereditary predisposition to breast cancer as well as uncovering of the molecular and mechanistic details of how the defective gene function increases the cancer risk of heterozygous carrier individuals. A central goal of this activity, based on the accumulated new information, is eventually to establish some guidelines and recommendations for the clinical utilisation of PALB2 mutation data.

Last updated: 10/5/2017