Abstract:
Breast cancer has been identified as the most common malignancy among
Pakistani women, accounting for 34.6% of all female cancers. Around 80-90%
cases are sporadic in nature. Multiple factors, including genetic as well as
epigenetic factors are responsible for the initiation of breast cancer. In the present
study, we carried out genetic characterization of breast cancer in our local
population. Diagnosed breast cancer patients having a mean age of 53yrs were
included in this study. These patients were analyzed for mutations in BRCA1 and
two metastasis suppressor genes (KAI1 and Kiss1). Germline mutations of three
genes BRCA1, Kiss1 and KAI1 were investigated in already diagnosed sporadic
breast cancer females. BRCA1, a tumour suppressor gene is found responsible for
majority of breast cancer cases of familial history. However its involvement in
sporadic breast cancer cases (especially of Pakistani population) is still an area
that requires further research. BRCA1 proteins binding domain encoding regions
were screened in this regard. Five silent mutations along with one novel splice
site mutation were observed on BRCA1. However, most of the BRCA1 portions
analyzed remained conserved at genomic levels. This study thus indirectly
highlights the involvement of other signaling cascade molecules as well as
transcriptional and translational regulatory mechanism responsible for breast
cancer.
Metastasis is among one of most lethal attribute of cancer. In this study, two
metastasis suppressor genes (Kiss1 and KAI1) were screened for any germline
mutations/polymorphism in these patients. So far no previous findings with
respect to these genes have been reported. In our study no exon or introns
deletions had been observed on these genes. Altered expression (down-
regulation) of these genes has extensively been reported in the literature in
relation to different types of cancers. However, expressional correlation of KAI1
with disease progression is established for the first time by this study. Significant
decrease in KAI1 expression has been observed in localized and invasive cancer
cells when compared to normal cells. Significant correlation of KAI1 with TNM
staging (Tumour, node and metastasis) has also been established (p=0.045).Patients showing higher expression of KAI1 showed better survival rate (after
median follow up of 120 months) when compared to those showing less or almost
negative expression (p=0.0136). Thus potential involvement of KAI1 molecule as
a marker of prognostic significance is established.
Interaction of KAI1, tetraspanin family member, with FAK, Shh, EGFR and
integrins was done as preliminary screening to find its potential involvement in
the metastatic cascade. A chain of in vitro assays were conducted to explore
KAI1 cellular response. Two transgenes based effective knock outs (MDA-MB-
231 KAI1KO1 , MDA-MB-231 KAI1KO2 ) and one forced expression (MCF-7 KAI1Exp1 )
were successfully generated through the aid of molecular tools. Expression levels
of KAI1 in both MDA-MB-231 and MCF-7 were confirmed through RTPCR and
western blot. No significant association of KAI1 on cancer cells growth had been
observed. This shows that KAI1 does not influence cell proliferation. However, a
direct relationship of KAI1 with intracellular attachment to basement membrane
has been established in both knock out as well forcedly expressing breast cancer
cell lines. Increased expression of KAI1 resulted in strong cells adhesion to
basement membrane in MCF-7
KAI1Exp1
when compared to both wild type and
controls (MCF-7 Wt , MCF-7 Ct ) with p value of 0.021. Similarly weak adhesion of
cancer cells has been observed in MDA-MB-231 KAI1KO1 and MDA-MB-231
KAI1Ko2
when compared with MDAMB-231 Wt and MDA-MB-231 Ct ) with p values
of 0.002 and 0.0004 respectively. A significant increase in cancer cell invasion
was observed in both ribozymes based KAI1 knock outs (MDA-MB-231 KAI1KO1
and MDA-MB-231 KAI1Ko2 in relation to control (MDA-MB-231 Ct ) with p values
of 0.0063 and 0.007 respectively. Furthermore, MCF-7 KAI1Exp1 cells showed
reduced cell invasiveness in relation to MCF-7 wt and MCF-7 Ct (p=0.022). Cell
motility was also analyzed in both absence and forcedly expressing KAI1 cell
lines. MDA-MB-231
KAI1KO1
and MDA-MB-231
KAI1KO2
showed restricted cell
migration (p=0.003 vs control) towards the wound. Increased cellular migrations
in time lapse based video (up to 90min) recording of MCF-7
KAI1Exp1
was
observed (p-0.024 vs control).
This study has shown that both genetic and epigenetic factors are mainly
responsible for systematic progression. Although the contributory role of gene
mutations is low but expressional regulation of these genes is an area which can
help in identifying the missing links. Establishing a marker of prognosticsignificance as well as its potential role in metastatic cascade suppression can
help in designing most promising gene therapy for breast tumour affected
patients.