Protein kinase Cδ expression in breast cancer as measured by real-time PCR, western blotting and ELISA

Abstract

The protein kinase C (PKC) family of genes encode serine/threonine kinases that regulate proliferation, apoptosis, cell survival and migration. Multiple isoforms of PKC have been described, one of which is PKCd. Currently, it is unclear whether PKCd is involved in promoting or inhibiting cancer formation/progression. The aim of this study was therefore to investigate the expression of PKCd in human breast cancer and relate its levels to multiple parameters of tumour progression. Protein kinase Cd expression at the mRNA level was measured using real-time PCR (n ¼ 208) and at protein level by both immunoblotting (n ¼ 94) and ELISA (n ¼ 98). Following immunoblotting, two proteins were identified, migrating with molecular masses of 78 and 160 kDa. The 78 kDa protein is likely to be the mature form of PKCd but the identity of the 160 kDa form is unknown. Levels of both these proteins correlated weakly but significantly with PKCd concentrations determined by ELISA (for the 78 kDa form, r ¼ 0.444, Po0.005, n ¼ 91 and for the 160 kDa form, r ¼ 0.237, P ¼ 0.023, n ¼ 91) and with PKCd mRNA levels (for the 78 kDa form, r ¼ 0.351, P ¼ 0.001, n ¼ 94 and for the 160 kDa form, r ¼ 0.216, P ¼ 0.037, n ¼ 94). Protein kinase Cd mRNA expression was significantly higher in oestrogen receptor (ER)-positive compared with ER-negative tumours (P ¼ 0.007, Mann -Whitney U-test). Increasing concentrations of PKCd mRNA were associated with reduced overall patient survival (P ¼ 0.004). Our results are consistent with a role for PKCd in breast cancer progression.

The role of S100 genes in breast cancer progression

“…The median followup for these patients was 41.8 months (range, 7.9-60 months). The storage and processing of the tissues were as previously described [9]. The second (cohort II) involved patients from a previously described and publicly available database [10].…”

“…The primer sequences, reaction mixture, and optimized amplification protocol for each of the housekeeping genes were as previously described by Mc Kiernan et al [9] Quantitect® primer kits from Qiagen (Hilden, Germany) were used for S100 expression analysis. Each reaction mixture contained 2 μl of cDNA, 2 μl of each Quantitect® primer and 10 μl of Quantitect® SYBR green reaction master mix in a final volume of 20 μl (Qiagen and Roche Diagnostics GmbH).…”

Protein kinase C: perfectly balanced

“…Reyland and colleagues have shown that elevated PKCδ mRNA levels negatively correlate with prognosis in Erb2-positive breast cancer, with mouse models suggesting that it is required for ErbB2-driven mammary gland tumorigenesis (Allen-Petersen et al 2014). Elevated PKCδ mRNA has also been reported to correlate with poor survival outcome in estrogen receptor-positive breast cancer (McKiernan et al 2008; Gyorffy et al 2010). Establishing whether PKC isozymes may play oncogenic roles in specific contexts in specific cancers, such as breast cancer, awaits functional characterization of mutations in PKC isozymes in these cancers (Garg et al 2014).…”

Differential subcellular expression of protein kinase C betaII in breast cancer: correlation with breast cancer subtypes

“…High PKCd expression correlated with endocrine responsiveness, while coexpression of PKCd and a was predictive of endocrine resistance in advanced breast cancer [10]. In other cohorts of breast cancer, PKCa was downregulated in advanced tumor samples [11], while elevated PKCd mRNA levels were associated with poor outcome [12]. PKCe was shown to be a predictive biomarker of aggressive breast cancer as well as a potential therapeutic target [13].…”