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  • GW311616 br The association between PKC activity and cancer progression leading


    The association between PKC activity and cancer progression leading to metastasis is well established [55,66,67]. Enhanced aggressiveness has been associated with a loss of E-cadherin and alteration in β-catenin expression [23,26,68]. In an earlier study, we showed that a number of EMT-related genes including E-cadherin were significantly downreg-ulated when MCF7 GW311616 overexpressing claudin 1 were compared to 
    control cells [33]. However, primers for β-catenin were not included in the commercial PCR array platform used for those studies. Thus, in this study, Western blot analysis to determine the effect of TPA treatment on the regulation of the β-catenin protein was conducted.
    A modest, however significant, downregulation of β-catenin protein upon TPA treatment was observed (Figure 8). In this same study, E-cadherin was also modestly but significantly downregulated in response to TPA treatment. This downregulation of E-cadherin in response to increased claudin 1 levels was consistent with our previous studies [33].
    PKC and ERK Inhibitors Block Claudin 1 Upregulation
    570 Claudin 1 Is Highly Upregulated by PKCBlanchard et al. Translational Oncology Vol. 12, No. 3, 2019
    Claudin 1 PKCε
    Figure 10. Representative immunostaining of ER+ and ER− breast tumors with the claudin 1, and PKCε antibodies. Staining was specific for tumor tissue, and while both membrane (black arrows) and cytoplasmic staining (red arrows) were observed with the claudin 1 antibody, staining with the PKCε antibody was confined to the cytoplasm (red arrows). Scale bar = 25 μm.
    Figure 11. Kaplan-Meier graphs for disease free survival in the breast tumor cohort. Univariate survival analyses were performed using Cox regression. Symbols on the graph lines represent censored data (recurrence or death from the disease). ER+ group n=447; events=
    Claudin 1 Is Highly Upregulated by PKC Blanchard et al. 571
    Table 1. Comparison of the Clinical-Pathological Characteristics of the Breast Tumor Cohorts.
    Patient age
    Median 65
    Tumor grade # cases % # cases % # cases % # cases %
    Nodal status
    Tumor size
    Claudin 1 (H-score) # cases median # cases median # cases median # cases median b.0001
    Recurrence (5 years)
    effectiveness in blocking claudin 1 upregulation by TPA. Go6976, which is known to inhibit PKCα and PKCβ, only partially inhibited claudin 1 upregulation, whereas rottlerin, the PKCδ inhibitor, appeared to have no effect. However, the PKC inhibitor GW311616 GF109203, which is known to inhibit PKCα, β, δ, and ε, significantly inhibited claudin 1 induction by TPA in the MCF7 cells (Pb.05) as shown in Figure 9, A and B, suggesting that PKCε may be involved in the upregulation of claudin 1.
    We further demonstrated that upregulation of claudin 1 in MCF7 cells was blocked by an ERK inhibitor but not a JNK inhibitor (Figure 9, B and C). Interestingly, in the MDA-MB231 cell line, although the ERK and JNK pathways were also activated by PKC, the upregulation of claudin 1 was not blocked by any of these inhibitors (Figure 9D).
    Correlation of PKCε with Claudin 1 in Human Invasive Breast Cancer Biopsies
    Based on the observations suggesting that PKCε may play a central role in the PKC/claudin 1 pathway, TMA analysis was carried out on a large cohort of human breast tumor patient biopsies to examine whether there was an association between PKC isomers and claudin 1 (Table 2, Figure 10). The cohort was comprised of ER− (basal and nonbasal) and ER+ human breast tumors (a total of 768 biopsies; see Materials and Methods.). 
    We observed a significant correlation between PKCε and claudin 1 in both the ER+ and ER− tumors (basal-like and non–basal-like) subgroups in this cohort (Table 2). Interestingly, we also found a significant positive correlation between claudin 1 and PKCα in ER+ tumors (Table 2).