Scott, Hannah Elizabeth
(2012)
PKC-δ, its C2 domain and breast cancer cell lines.
PhD thesis, University of Nottingham.
Abstract
Protein kinase C δ (PKC-δ) is a novel member of the PKC family of serine-threonine kinases. PKC-δ structure is widely conserved within the PKC family and has a catalytic region and regulatory region. The regulatory region has two main sub domains C1 and C2. Although several studies have investigated the role of the C1 domain, little is known about the function of the C2 domain, however there is some evidence that it acts as a protein interaction domain.
PKCs are involved in a wide variety of cellular functions within cancer. PKC-δ has been demonstrated to have a particular involvement with the apoptotic processes of a cancer cell. A pro-apoptotic role for PKC-δ has been identified, whereby tyrosine phosphorylation of particular residues induces translocation to the nucleus, alongside a similar translocation event for caspase-3. In the nucleus, caspase-3 cleaves the regulatory and catalytic regions to form a free catalytic domain that is uninhibited by the regulatory portion. This free catalytic region causes the induction of the apoptotic pathway. Conversely an anti-apoptotic role has also been identified for PKC-δ. This was found in MDA-MB-231 cells, which have a ras mutation. Due to this mutation in these cells, ERK1/2 phosphorylation is high. Without PKC-δ activity the high phosphorylation levels induce apoptosis. PKC-δ acts on a pathway to reduce ERK1/2 phosphorylation, thus facilitating cell survival.
This study aimed to investigate the role of the PKC-δ C2 domain within breast cancer cells. Through use of these techniques, the role of the C2 domain was examined in order to consider its utility as a drug target to treat breast cancer.
•Constructs were developed of pIRESneo2 vector with myc-tagged C2 domain and myc-tagged PKC-δ sequences
•Breast cancer cell lines, MDA-MB-468, MDA-MB-231 and MCF-7, were stably transfected with a vector control and myc-δC2 construct. MDA-MB-231 and MCF-7 were also transfected with myc-PKC-δ.
•Cell lines developed were examined for alterations due to the presence of the C2 domain or PKC-δ over-expression
As the C2 domain is proposed to be a protein interaction domain, we hypothesized that over-expression of this domain would interfere with endogenous PKC-δ interactions, through competitive inhibition, and thus we could identify C2 domain roles. The effect on the cells of the endogenous PKC-δ would be opposed by the C2 domain. Thus the role of endogenous PKC-δ could also be clarified for a particular situation - it would be the opposite of the effects induced by the myc-δC2 on the cells.
In the MDA-MB-468 stable cell lines, immuno-fluorescence examination of the myc-δC2 cells showed the myc-δC2 was localised at the ends of actin protrusions from the bulk of the cell. The myc-δC2 expressing cells had a more extensive cytoskeleton than the Vector control cells, possibly suggesting improved attachment to a surface. An experiment examining this illustrated that the myc-δC2 cells appeared to attach in a shorter time period. This implies that the role of the endogenous PKC-δ is to discourage cell attachment and promote an invasive phenotype, this is in agreement with the literature.
During sub-culture of the MDA-MB-468 cell lines it became apparent that the myc-δC2 cells were growing at an increased rate over the Vector cell lines. This was quantified and indeed the myc-δC2 cells did increase in cell number more than the Vector cells. This was also the case with the MDA-MB-231 cells but not with the MCF-7 cells. Growth is a balance of proliferation and apoptosis. This effect indicates that PKC-δ is pro-apoptotic, or anti-proliferative. MCF-7 cells lack caspase-3 and thus pro-apoptotic effects of PKC-δ would be affected in this cell line. As the myc-δC2 did not have an effect in these cells we examined apoptosis to see if these effects could be attributed to differences in apoptosis.
The MDA-MB-468 cells expressing myc-δC2 had higher viability than the Vector cells. This fits with the cell number data, indicating that a lower level of apoptosis has led to a greater cell number, and advocates a pro-apoptotic role for PKC-δ. This was not the case in MDA-MB-231 cell lines where Vector cells had higher viability. This agrees with the literature describing PKC-δ displaying an anti-apoptotic role in this cell line, but does not fit with the cell number data. Thus, differences in cell number are likely due to effects on proliferation, although this was not investigated. MCF-7 cells showed no differences indicating the apoptotic program of these cells is indeed affected by the lack of caspase-3.
Serum starvation is a commonly used method to induce apoptosis. MDA-MB-468 cell lines were serum starved in order to examine the effects. The apoptosis profile was altered and myc-δC2 cells showed lower viability than the Vector cells, indicative of an anti-apoptotic effect of PKC-δ. An anti-apoptotic effect is observed in MDA-MB-231 cells, where the effect was proteasome dependent. This was also the case in this situation. The anti-apoptotic effect is related to levels of phosphorylated ERK1/2, where high levels are pro-apoptotic. The phosphorylation status was examined and illustrated a much higher level of phosphorylation in myc-δC2 cells over Vector cells when starved. This indicates myc-δC2 is inhibiting the de-phosphorylating role of PKC-δ in these apoptotic cells. Thus it appears that the C2 domain acts as a ‘sensor’ to serum status, appropriating PKC-δ effects in apoptotic pathways according to the serum status, the method of which is unknown.
This study has highlighted the importance of the PKC-δ C2 domain in breast cancer apoptosis. The effects appear to require a fully active PKC-δ pro-apoptotic pathway, and are dependent on the serum status of the cells. Further investigation would be required to identify a level of serum for use in vitro that is relevant to an in vivo tumour situation. If low levels are more relevant to a clinical state then it may be possible to target the C2 domain with drugs to allow induction of apoptosis. The differences between the cell lines clearly show that the phenotypic analysis of tumours would be vital to identifying whether such treatment would be applicable, as effects of any drug would vary greatly across tumour types. MDA-MB-468 and MDA-MB-231 are both triple negative cell lines (i.e. they do not express progesterone, oestrogen or HER2 receptors); however the strong differences seen in this case indicate further phenotypic analysis would be essential.
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