3.1. Knock down of ZEB1 in invasive cancer cells causes re-expression of PKP3
Deconstruction of junctional complexes allows differentiated epithelial cells to convert to a motile fibroblastoid phenotype during embryonic development and physiological events in the adult organism [13
]. This process, generally termed epithelial-mesenchymal transition (EMT), is also pathologically activated during cancer cell invasion and metastasis [15
]. Recently, we have identified the transcription factor ZEB1 (δEF1, TCF8, AREB6) as a direct transcriptional repressor of E-cadherin and a potent inducer of EMT in human breast cancer cells [11
] (see also ). In addition, depletion of ZEB1 in dedifferentiated and metastatic cancer cells by RNA interference not only restored E-cadherin expression but also caused the reestablishment of epithelial features including the partial formation of adherens and tight junctions [11
]. These data suggest that ZEB1 may regulate various genes critically involved in the formation of epithelial adhesion complexes.
Fig. 1 Knock down of ZEB1 causes upregulation of PKP3 expression. (A) RNAi-mediated knock down of ZEB1 in MDA-MB-231 breast cancer cells. Three days after treatment with either ZEB1-specific (si-ZEB1) or unspecific scrambled (si-Control) siRNAs total RNA was (more ...)
In order to determine the function of ZEB1 during cancer progression, we specifically knocked down ZEB1 in invasive and dedifferentiated MDA-MB-231 cancer cells expressing high levels of endogenous ZEB1 and lacking all junctional adhesion complexes, and identified de-repressed genes by Affymetrix Gene Chip™ analyses. Among the genes whose transcript levels were significantly upregulated upon ZEB1 depletion, we detected the desmosmal protein PKP3 (). RT-PCR experiments using cDNA samples from three independent siRNA knockdown experiments confirmed the PKP3 Gene Chip data. In all experiments only low amounts of PKP3 transcripts were detected in control cells treated with unspecific scrambled siRNAs, whereas strong expression was observed upon specific depletion of ZEB1 (, one representative example is shown). Next we analysed whether the increase in PKP3 transcripts concomitantly led to the expression of functional protein products. MDA-MB-231 cells were treated with unspecific or ZEB1-specific siRNAs for three days and total cell lysates were probed for PKP3 protein by immunoblotting. Only minor amounts of PKP3 were detected in MDA-MB-231 control cells, whereas ZEB1 depletion caused strong upregulation of PKP3 protein levels ().
Immunofluorescence microscopy confirmed that PKP3 was upregulated in ZEB1-depleted MDA-MB-231 cells and located to spot-like structures in the cytoplasm or at the lateral plasma membrane (). In contrast, control cells exhibited only weak and diffuse cytoplasmic stainings (). However, defined dotted staining patterns along the whole lateral membrane, which are typical for desmosomal structures in fully polarized epithelia, could not be observed. Similar observations were made regarding the expression and intracellular localization of the desmosomal plaque protein Desmoplakin (). The lack of fully differentiated desmosomes might be due to the short-term inhibition of ZEB1 in the transient siRNA experiments (3 days siRNA treatment). At this stage the tight junction marker ZO1 only partially accumulated at the plasma membrane (). Alternatively, critical desmosomal components might still be missing and/or limiting in ZEB1-depleted MDA-MB-231 cells preventing complete assembly of desmosomes. Taken together, the knock down of ZEB1 caused strong derepression of PKP3 on both the mRNA and protein level and caused partial relocalization of PKP3 to the lateral plasma membrane.
Fig. 2 PKP3 is partially relocalized to the plasma membrane upon ZEB1 knock-down. For immunofluorescence microscopy MDA-MB-231 cells were treated with ZEB1-specific (si-ZEB1) or unspecific scrambled (si-Control) si-RNAs for three days, fixed with formaldehyde (more ...)
3.2. Expression of E-cadherin is not sufficient to induce PKP3 expression
Besides its major structural role in epithelial cell-cell adhesion E-cadherin can critically influence intracellular signalling pathways such as Wnt and receptor tyrosine kinase signalling [17
]. Moreover, many reports demonstrated that ectopic E-cadherin expression is sufficient to confer epithelial differentiation to fibroblasts and dedifferentiated cancer cells [18
]. As re-expression of functional E-cadherin is one of the hallmarks of ZEB1 knock down in MDA-MB-231 cancer cells [11
], we investigated whether E-cadherin expression may be sufficient to induce PKP3 expression in a ZEB1 independent manner. We generated MDA-MB-231 cells stably expressing GFP-tagged E-cadherin, which has been shown previously to be functional and confer adhesion to E-cadherin-negative mesenchymal cells [19
]. Ectopically expressed E-cadherin was enriched at sites of cell to cell contacts but accumulated also in the cytoplasm (Supplementary Fig. 1A). However, the cells did not display polarized and differentiated epithelial features, but formed only irregularly structured and weakly adhesive cell clusters (Supplementary Fig. 1A). In these cells PKP3 expression was induced neither at the mRNA nor at the protein level (Supplementary Fig. 1A and B). Expression of E-cadherin also did not alter ZEB1 expression levels in MDA-MB-231 cells (data not shown). Thus, expression of E-cadherin alone was not sufficient to upregulate PKP3 transcript or protein levels.
3.3. ZEB1 physically associates with the PKP3 promoter and represses its activity
Next we analysed whether PKP3 represents a direct bona fide
target gene of ZEB1 in MDA-MB-231 cancer cells. ZEB1 was previously shown to specifically interact with E-box elements in the proximal promoter region of target genes such as E-cadherin [11
]. Hence, we searched the human and mouse proximal PKP3 promoter regions for the presence of E-box elements (5′-CACCTG-3′). Within the first 4 kbp upstream of the PKP3 transcription initiation site we detected six and seven E-box consensus sites in the human and mouse promoter, respectively, of which the two E-box elements closest to the initiation of transcription were highly conserved between both species (; ; Supplementary Fig. 2). On the other hand two additional highly conserved stretches located further upstream did not contain E-box elements conserved in human and mouse (Supplementary Fig. 2). Hence, we performed chromatin immunoprecipitation (ChIP) assays to investigate whether ZEB1 can directly associate with the conserved E-box elements in the human PKP3 promoter in vivo. ZEB1-associated chromatin was immunoprecipitated from human MDA-MB-231 cells that express high levels of endogenous ZEB1 [11
] using an antibody specifically recognizing the N-terminal domain of ZEB1 [11
]. Chromatin fragments associated with ZEB1 were analysed for the presence of PKP3 promoter sequences by PCR using primers that either span the two conserved proximal E-boxes (−143/+43; ChIP1) or E-box 6 located further upstream (−3800/−3670; ChIP2) in the human PKP3 promoter (). As depicted in the ZEB1 antibody consistently pulled down chromatin fragments containing the two proximal E-boxes elements, whereas fragments containing the most upstream E-box could not be detected. In addition, unspecific control antibodies did not precipitate any PKP3 promoter sequences ().
Fig. 3 ZEB1 directly associates with the PKP3 promoter and represses its activity. (A) Scheme of the human PKP3 promoter. Relative positions of E-boxes are depicted as black bars along the first four kbp of the human PKP3 promoter. (B) The two proximal E-boxes (more ...)
Fig. 4 ZEB 1 represses the activity of the mouse PKP3 promoter. (A) Scheme of the first four kbp of the mouse PKP3 promoter. E-boxes are indicated by black bars. The position of the four promoter fragments are shown below the scheme (mPKP3-prom1-4). (B) Reporter (more ...)
To determine whether binding of ZEB1 to the proximal PKP3 promoter region in vivo is sufficient to repress the human PKP3 promoter activity, we generated two different but overlapping luciferase reporter constructs (−496/+43, hPKP3-prom1 and −934/+43, hPKP-prom2), both containing the proximal conserved E-boxes of the human promoter. These constructs were transfected into human MCF7 epithelial cells, which express high levels of endogenous PKP3 (Supplementary Fig. 1), together with a human ZEB1 expression vector or a control vector expressing an unrelated protein (p120ctn) as well as a β-galactosidase vector for normalization of transfection efficiency and viability. Compared to the control vector, expression of ZEB1 caused a significant repression of both human PKP3 promoter constructs (). Similar to ZEB1 the transient overexpression of the E-box binding repressor Snail1 also interfered with the activity of the human PKP3 promoter.
In order to investigate whether the activity of the mouse PKP3 promoter is also affected by ZEB1, we tested four different mouse PKP3 promoter constructs in reporter gene assays (). Two proximal shorter fragments encompassing the regions −411/−2 (with only the two conserved E-boxes; named mPKP3-prom1) or −877/−2 (with three E-boxes; named mPKP3-prom2), and two longer fragments ranging from −3177 to −2 (with six E-boxes; named mPKP3-prom3) and −3808 to −2 (with seven E-boxes; named mPKP3-prom4) were analysed in mouse mammary epithelial cells (EpH4) as described above using a full-length mouse ZEB1 expression construct [11
]. Albeit ZEB1 repressed the activity of all four PKP3 promoter regions, repression was particularly effective with the two shorter reporter constructs containing mainly the proximal conserved E-boxes (). These data indicate that in analogy to the human PKP3 promoter, the proximal E-boxes were necessary and sufficient to mediate repression by ZEB1. Only weak repressive effects were observed for the longer constructs (). Hence, the longer promoter fragments might additionally contain regulatory elements that partially relieve the repressive effects of ZEB1. Taken together, our data demonstrate that endogenous ZEB1 can directly associate with the proximal PKP3 promoter in undifferentiated cancer cells and partially repress both human and mouse PKP3 promoter activities. Repression is mediated by conserved proximal E-box elements close to the transcription initiation site.
3.4. ZEB1 represses PKP3 during cancer cell invasion in vivo
To determine whether ZEB1 is able to repress PKP3 expression during cancer cell invasion in human tumours we performed double-immunohistochemical analyses of 10 paraffin-embedded human colon cancer specimens using antibodies to PKP3 and ZEB1. Colon cancer represents a suitable cancer type to study ZEB1 function in vivo, as ZEB1 has already been implicated in the repression of basal lamina components in colon tumours [23
Immunohistochemistry revealed that within the differentiated bulk tumour area PKP3 localized primarily to the plasma membrane of cancer cells (, violet stain, arrows). Accordingly, these cells express high amounts of E-cadherin and cytokeratin emphasizing their differentiated epithelial phenotype (data not shown). These differentiated cancer cells completely lacked detectable ZEB1 expression (, no brown stain in cells indicated by arrows). However, ZEB1 accumulated in the nuclei of a large number of tumour-associated stroma cells (, brown stain, arrowheads), whereas in normal colon stroma ZEB1 positive cells were only rarely detected (data not shown). Within the stroma ZEB1 might be specifically expressed in fibroblasts and partially in activated fibroblasts expressing Smooth Muscle Actin (SMA) (, SMA, violet; ZEB1, brown. Arrowheads indicate colocalization of SMA and ZEB1). At present one can only speculate whether some of the ZEB1 positive stromal fibroblasts might have directly developed from epithelial cancer cells through a bona fide
ZEB1-dependent EMT (compare also with [32
Fig. 5 ZEB1 and PKP3 localization in human colon cancer. (A) Colon cancer specimens were double-stained for ZEB1 (brown stain) and PKP3 (violet stain). Left panel, overview of differentiated bulk tumour area. Right panel, higher magnification of differentiated (more ...)
Colon tumours showed defined areas of strong invasion, where loosely attached or single cancer cells invade the underlying stroma (, Hematoxylin/Eosin stain of invasive colon cancer area). In contrast to the bulk tumour area, ZEB1 was strongly upregulated in distinct invading cancer cells at the tumour-host interface. Upregulation of ZEB1 was clearly visible in eight out of 10 cancer specimens (, brown stain, arrowheads). Based on nuclear and cellular morphologies the ZEB1-positive cancer cells were clearly different from the ZEB1-expressing stroma cells (compare arrowhead marked cells in ). ZEB1 expression was always accompanied by defects in intercellular adhesion and loss of membrane associated PKP3. However, the cells still exhibited a rudimentary cytoplasmic PKP3 pool, which directly proved their epithelial origin ().
In conclusion, significant levels of membrane-bound PKP3 can only be found in the differentiated main tumour mass of colorectal cancers barely expressing any ZEB1, whereas upregulation of ZEB1 at the invasive front was accompanied by downregulation of PKP3 protein levels and loss of intercellular adhesion.