Studying the Genetic Basis of Sporadic Breast Cancer Metastasis

Abstract

Metastasis is the most lethal attribute of breast cancer and is responsible for majority of cancer related deaths. Numbers of metastasis suppressor genes that act to prevent or control metastasis have been identified in several types of cancers including breast cancer. This study was designed to screen three metastasis suppressor genes (DRG1, PTEN and gelsolin) for germ line mutations in sporadic breast cancer cases of Pakistani population. For expressional studies these genes were screened in both Pakistani as well as in British cohort. Three different study groups were recruited for this study. Cohort 1 was from Pakistani population, comprised of 350 blood samples and used for mutational analysis by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP). Amplified products showing altered mobility patterns were sequenced and analyzed. For mRNA expression analysis, two different cohorts, (cohort 2 & cohort 3) from two different populations were used. Cohort 2 was from Pakistani population and comprised of 60 tissue samples while tumor matched normal adjacent tissues were used as control. Cohort 3 was from British population and comprised of 114 breast cancer tissues and 31 normal background tissues. Cohort 2 and 3 were screened by using real time quantitative polymerase chain reaction (qPCR) and data obtained was compared according to patients’ outcome and survival. Additionally, DRG1 expression was targeted in vitro using ribozyme transgene technology to explore the function of DRG1 in two human breast cancer cell lines. In this study using cohort 1, mean age of patients was calculated as 47±1.1 years, age at menarche as 13±0.1 years and mean age of post menopausal patients at menopause was calculated as 45±0.5 years. After Single Strand Conformational Polymorphism (SSCP), 57 samples showing altered mobility patterns were observed in DRG1. Splice site non-synonymous substitution and frame shift mutations were observed on exon 1 and exons 3, 4, and 5 respectively. Overall, clear majority of the sequenced mutations, 51 mutations out of 57 were frame shift mutations, which were deletions and 6 were splice site substitution mutations. Deletion mutation 459A/- was detected with highest frequency among all variations (0.36). Most prevalent mutation among premenopausal patients was frame shift mutations which were 369A/-, 377A/-, 387A/- in exon 4. While frame shift mutation 459A/- in exon 5 was most prevalent among postmenopausal patients. When analyzed according to age group frame shift xmutations in exon 4 (369A/-, 377A/-, 387A/-) was found most common in age <40 years. While 459A/- in exon 5 was wide spread among patients >40 years of age. It was observed that DRG1 expressed aberrantly in clinical breast tumor tissues in both cohorts 2 and 3 and was found to be significantly down regulated compared to control tissues. A highly significant link was seen between low levels of DRG1 expression and metastatic development. Patients who died of breast cancer also showed significant down regulation of DRG1 in cohort 3. Sequencing analysis revealed nineteen different types of mutations in different regions of PTEN (in exons 2, 4, 5, 6, 7 and splicing sites of intron 2 and 4 and also in the 3’ UTR region), including 3 silent, 8 missense, 2 frame shift and 6 splice site variations. Among the observed variations in this study, three missense mutations have already been reported i.e. 319G>A (Asp106Asn), 389G>A (Arg129Gln) and 482G>A (Arg160Lys) in different populations. Substitution at 3’ UTR region of PTEN 2634 T>A was observed with a highest frequency (0.139). Most common mutation in premenopausal patients was frame shift mutation that was -/A in exon 7. While substitution mutation T>A in 3’UTR region was most prevalent among postmenopausal patients. Most prevalent mutation among patients in age group <40 years was substitution mutation that was T>A in 3’UTR. While -/A, mutation in exon 7 was most prevalent in patients >40 years of age. Significant low levels of PTEN were observed in low grade tumor and in patients with poor prognosis in cohort 2. No significant difference was observed in transcript levels of PTEN, when analyzed according to grades, NPI value and TNM staging in cohort 3. Different types of mutations were observed in gelsolin which include 3 non- synonymous substitutions, 1 synonymous substitution and 10 frame shift mutations (comprising of 4 duplications, 5 deletions and 1 insertion) that were located in exons 4, 6, 7, 8, 10, 11, 14. Insertion mutation observed in exon 10 in 36 samples has highest frequency (0.116) among all mutations observed. Most common mutation found in premenopausal patients was frame shift mutation that was 539 A/- in exon 7. Frame shift mutation 897_898------/GCAGGC in exon 10 was most prevalent among postmenopausal patients. This variation was also the most prevalent mutation among patients in age group <40 years. While 987_988 C>T, 987_988 --/TC in exon 11 was wide spread among patients >40 years of age. Negative correlation of gelsolin xitranscript levels with development of metastasis in breast cancer patients in both cohorts (2 and 3) was observed. In vitro study revealed that knockdown of DRG1 results in significantly increased invasion and motility and decreased matrix-adhesion in MCF-7 cells. In this study different risk factors were also analyzed in association with breast cancer in patients but no association of these factors was observed in Pakistani population which is in accordance with many earlier studies conducted in this population. A wide range of germline mutations observed in DRG1, PTEN and gelsolin were found to be in important domains and might impair their functional activities. It can be suggested that these variations may play an important role in the pathogenesis and progression of breast cancer metastasis. Low expressional levels of these genes appear to be linked to development of metastasis and may be useful as a prognostic factor. In vitro data presented here indicate an involvement of the DRG1 gene in breast cancer progression and demonstrate a potential role of this gene in suppressing tumor metastasis.