To develop effective therapies that stop breast cancer from metastasising, the underlying biological and molecular events need to be understood in further detail. We show here that the PKD family members PKD1, PKD2 and PKD3 are all expressed in ductal epithelial cells of the normal breast (Figure ). We further show that decreased expression of PKD1 can serve as a marker for invasive breast cancer, whereas PKD2 and PKD3 expression remain unchanged in normal breast and invasive breast tumour tissue (Figure ). However, all three PKD enzymes are markers for breast epithelial cells (normal and tumour) and may be utilised as markers to identify breast epithelia-derived metastases. PKD1 expression was downregulated by approximately 60% in more than 95% of the analysed samples of invasive ductal carcinoma and distant lymph node metastases (Figure ). All 50 analysed tumours were assessed by pathologists and stages were at a range from 0, IIA, IIB, IIIA, IIIB, IIIC. Further, additional information such as sex, age, diagnosis, pTNM, lymph node stage (positive lymph nodes/examined lymph nodes), as well as expression of the PR, or the more-aggressive ER-negative, basal sub-type of breast cancer were available. Downregulation of PKD1 expression occurred in more than 95% of the analysed cases of invasive ductal cancer and no correlation was observed with stage, ER, PR or other markers. Our results on PKD1 in invasive breast cancer are in consensus with data obtained for gastric cancer and prostate cancer, where decreased expression of PKD1 was described in most of the cases analysed [14
Our data showing reduced PKD1 protein expression in invasive breast cancer is also in consensus with published transcriptional microarray data profiling over 350 surgically excised, advanced breast tumour tissues. In these arrays PRKD1
gene expression was drastically reduced in most cases analysed [41
]. Our data show that reduced gene expression invariably translates to decreased protein levels. Investigation of other publicly available microarray datasets on the NCBI Gene Expression Omnibus (GEO) showed that PRKD1
is detected at appreciable levels in normal lobular and ductal breast cells [GEO:GDS2635] [45
], in atypical hyperplasia [GEO:GDS1250] [46
] and in the cancerous lesions invasive ductal and lobular carcinomas [GEO:GDS2635] [45
], suggesting that PKD1 expression is indeed decreased with increased invasiveness of the tumours.
Little is known about the role of PKD1 in regulating tumour cell migration and invasion, important processes that regulate both tumour expansion and metastasis. In order to investigate a potential role for PKD1 in cell invasion, we first compared PKD1 expression in very low-invasive and highly invasive breast cancer cell lines (Figures ) and found that from the three PKD family members only PKD1 showed a significant expression pattern associated with the invasive phenotype. PKD1 expression was absent in highly invasive breast cancer cell lines including MDA-MB-231, T47D and SKBR3 (Figure ). This is most likely because of epigenetic silencing mediated by DNA methyltransferases (Figure ). Non-invasive or very low-invasive breast cancer cell lines such as BT-474 or MCF-7 and the normal breast cancer cell line MCF-10A moderately expressed PKD1. Moreover, by analysing PKD1 expression in the 1-HMT-3522 breast cancer cell progression model, we found that the T4/2 clone which shows increased invasiveness as compared with the S1 clone also expressed less PKD1 (Figure ).
We utilised two breast cancer model cell lines, MCF-7 and MDA-MB-231, to investigate the role of PKD1 in cell invasion. MCF-7 and MDA-MB-231 cells express comparable amounts of PKD2 and PKD3, but differ in their expression of PKD1 (Figure ). The depletion of PKD1 in MCF-7 cells resulted in increased cell invasion in both 2D and 3D cell culture systems (Figure ).
On the other hand, the re-introduction of active PKD-1 in MDA-MB-231 cells impaired their invasive behaviour in 2D and 3D cell culture (Figure ). Notably, the knockdown of PKD1 in MCF-7 cells (Figure ) and the induction of constitutively active PKD1 in MDA-MB-231 cells had no significant effects on cell proliferation or cell death (data not shown). This is interesting, because one of the PKD family members, PKD3, was recently linked to increased tumour cell proliferation in prostate cancer [47
]. This implies that in different cancers the three PKD family members may have different functions. A similar phenomenon was recently demonstrated for the kinase Akt, which, depending on the isoenzyme expressed, contributes to breast tumour cell survival and proliferation, or blocks cell migration and invasion [48
]. Cell proliferation, survival and cell motility are not necessarily linked in cancer cells, and it is generally accepted in the field that proliferation and invasiveness are independent of each other.
Our data further suggest that PKD1 inhibits breast cancer cell invasion by regulating the expression of factors involved in the degradation of ECM. The invasion of MDA-MB-231 cells in Matrigel is dependent on MMPs. For example, MMP-2, MMP-7, MMP-9, MMP-11, MMP-13 and MMP-14 are known to enhance the invasiveness of MDA-MB-231 cells [37
]. We found that the expression of active PKD1 in MDA-MB-231 cells downregulated mRNA transcripts of MMP-2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14 and MMP-15 (Figure ). Thus, PKD1 decreased the expression of all MMPs so far implicated in the invasive phenotype of this cell line. The mechanism of how PKD1 regulates such a multitude of genes is not known yet. One explanation is that PKD1 may regulate a common element in the promoter of these MMPs. In this context histone deacetylases (HDACs) have been shown to regulate the expression of MMPs [30
]. PKD1 is known to be a negative regulator of HDACs [49
] and it is possible that PKD1 exerts its effects on all the MMPs via regulation of HDACs.
We did not observe any differences in the expression of MMP-1, MMP-8, MMP-16, TIMP1 or TIMP2 by PKD1. Further, the expression of MMP-3 was slightly increased by active PKD1. This is interesting, because MMP-3 has been previously shown to inhibit cell invasion of MDA-MB-231 [40
]. MMP-3 expression was associated with benign and early stage breast tumours but is frequently lost in advanced stage, aggressive breast disease [40
]. The events leading to the transition from a benign to a metastatic tumour are not fully understood, but are linked to ECM degradation and increased motility of cells. It is possible that loss of PKD1 expression and the resulting change in the expression of MMPs is part of the switch driving the progression from a benign to an invasive, malignant tumour.