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  • br In addition our studies

    2022-04-28


    In addition, our studies also demonstrated that, at the protein level, there was a small, nonetheless significant, decrease in both E-cadherin and β-catenin protein upon PKC activation in MCF7 cells. This inverse relationship between these EMT regulators was consistent with similar results described in the other cancers [18,21,59].
    Long-term PKC activation of MCF7 N,N-Dimethylsphingosine also resulted in significant growth inhibition in all breast cancer cell lines examined (Figure 8). Inhibition of MCF7 cell growth has been previously described by Fortino et al. [65] following a shorter period of TPA treatment, which these authors further demonstrated was attributed to prosurvival and antiproliferative properties of TPA in these cells.
    To further define the PKC isomers and signaling pathways involved in claudin 1 upregulation in MCF7 cells, we used inhibitors of PKC to block PKC activation by TPA. The pan-inhibitor GF109203, which inhibits PKCα, β, δ, and ε, was most effective in blocking claudin 1 upregulation by TPA, whereas rottlerin, which inhibits PKCδ, appeared to have had no effect. Go6976, which inhibits PKCα and PKCβ, was only partially effective. Importantly, these results pointed to PKCε and, to a lesser extent, PKCα as possible regulators of claudin 1 in MCF7 cells.
    Increased PKCα expression has been reported in ER− breast tumor samples [80], and PKCα has also been identified as a marker of poor prognosis independent of other factors [70]. Patients with PKCα-negative tumors were also shown to have better responses to endocrine therapy [80,81]. Furthermore, in vitro, PKCα has been shown to enhance migratory potential and promote more aggressive behavior in MCF7 [82] and MDA-MB231 cells [51,70].
    Similarly, an oncogenic role has frequently been assigned to PKCε in breast cancer, and it is considered a marker of aggressiveness [72]. PKCε has been shown to be essential for enhanced cell migration and invasion and promote breast cancer survival [71]. High expression levels of PKCε have also been shown to correlate with high tumor grade, HER2 expression, ER negativity, and poor survival in patients with breast cancer [83]. In vitro, PKCε has been shown to promote cell survival by inhibiting apoptosis of MCF7 cells and to promote EMT [72]. It has been demonstrated that when PKCε was overexpressed in the nonmalignant immortalized breast epithelial cell line MCF10A, these cells lost their phenotypic epithelial characteristics and became fibroblastic and spindly, increased cell migration, and were protected from anoikis [72].
    We demonstrated that the upregulation of claudin 1 by TPA was effectively blocked by PKC and ERK inhibitors, but not by JNK inhibitors, providing strong evidence that claudin 1 induction in MCF7 cells was through the activation of the ERK pathway. Interestingly, the inhibition of the ERK signaling pathway did not block the upregulation of claudin 1 in the MDA-MB231 cell line, leading us to suggest that these two cell lines may have different PKC signaling pathways. Indeed, Platet et al. [84] previously demonstrated that TPA increased the invasiveness of MCF7 cells but decreased the invasiveness of MDA-MB231 cells and attributed this to the ER status and abnormal TPA regulation of PKCα activity in the MDA-MB231 cells. Another plausible explanation is that the differences observed in the responses of these cells to the PKC inhibitors may reflect the extent of leakiness at their tight junctions. Sjo et al. [85] demonstrated that the leakiness or tightness of the membrane barrier of cells affects the response of claudin family members to PKC activation.
    We also investigated whether there was a link between PKCε, PKCα, and claudin 1 in a large cohort of breast cancer biopsies (768 
    samples) of mixed pathologies. We further divided these samples into ER+ and ER− subgroups. The ER− tumors were then yet again divided into basal and nonbasal since, in an earlier study, we identified a relationship between high claudin 1 expression and the basal-like subtype of breast cancers. In the present study, we found a correlation between claudin 1, PKCα, and PKCε in patient biopsies. Both PKCε and PKCα positively correlated with claudin 1 in the ER
    + tumors. We also identified a further significant correlation between claudin 1 and PKCε in ER− tumors and basal and non–basal-like
    subtype of tumors. (Table 2). Additionally, our analysis showed that overall PKCε levels and claudin 1 were higher in the ER− tumors compared to ER+ tumors (Table 3).
    Interestingly, we identified a negative association between patient’s survival and PKCε. High PKCε levels significantly correlated with
    decreased survival and/or recurrence rate in both the basal and nonbasal ER− tumors. However, claudin 1 alone, whether high or low, had no effects on patient survival.