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  • The manuscript provides mechanistic insights into the suppre

    2021-10-12

    The manuscript provides mechanistic insights into the suppression of EGFR, BCRP, PKM2, and other cancer biomarkers in response to pharmacological inhibition of GPR55 with MNF and CID (Fig. 7). Firstly, inactivation of ERK has been previously demonstrated following treatment of PANC-1 Tasquinimod with MNF [21], consistent with the suppression of the upstream c-Raf/MEK cascade. Our earlier work has established that GPR55 is a key target of MNF in PANC-1 cells and the human hepatocarcinoma HepG2 cells [21]. The fact that the effects of MNF can be replicated by CID further supports the notion that GPR55 is indeed a major player in the antitumorigenic actions of MNF. Thus, pharmacological inhibition of GPR55 has a clear negative impact on the MEK/ERK pathway-dependent phosphorylation and nuclear translocation of PKM2, thereby reducing pro-oncogenic gene expression. Our data also identifies a feed-forward loop in which GPR55 activation leads to increased EGFR and PKM2 expression, thus potentiating the EGFR-MEK/ERK-PKM2 effects on proliferation and MDR (Fig. 7) [14], [15]. Secondly, the proposed EGFR-MEK/ERK-PKM2 pathway does not completely explain the data obtained in this study as GPR55 antagonism with MNF and CID caused also a significant attenuation of the constitutive and GPR55-inducible increases in phosphoactive AKT levels. Sustained activation of the PI3K/AKT pathway is required for enhanced β-catenin transcriptional activity through phosphorylation of β-catenin by protein kinase A at Ser-522 [33] and the inactivation of GSK-3β via Ser-9 phosphorylation [34], [35]. AKT-mediated inhibition of GSK-3β activity leads to the upregulation of Pgp, MRP-4 and BCRP expression [8], [36]; conversely, impaired AKT activity (e.g., lower pAKT levels) promotes the ability of active, unphosphorylated GSK-3β to phosphorylate β-catenin at Ser33, a modification that leads to the ubiquination and subsequent proteolytic degradation of β-catenin [36]. The PI3K/AKT pathway has also been associated with the induction of HIF-1α [17]. Here, the pharmacological inhibition of GPR55 was associated with impaired activation of the PI3K/AKT signaling pathway, which led to the attenuated expression and nuclear accumulation of β-catenin and HIF-1α (Fig. 7). The effectiveness of chemotherapy in a broad spectrum of tumors is limited by a multitude of factors including transporter-mediated MDR [1], [2], [3]. For example, incubation of human-derived PancCa tumor cell lines with standard of care agents such as gemcitabine, 5-fluorouracil, and cisplatin results in an elevation of MDR protein expression in surviving cells [4] and MDR-based resistance to frontline agents in breast cancer therapy is a well-established fact [5], [6]. One clinical strategy to overcome MDR is to co-administer inhibitors of MDR transport with the objective of increasing intracellular concentrations of the chemotherapeutic agents and achieving required therapeutic exposure [27]. An alternative strategy is to modulate MDR protein expression by targeting select tyrosine kinases such as EGFR and HER2 [37] or the Wnt-3a/GS3K/β-catenin pathway [16]. The data from this study demonstrate that GPR55 inhibition may be an additional approach to the clinical treatment of MDR. In addition, while MDR proteins are generally assumed to be localized in the cellular membrane, MDR protein expression has been identified at subcellular sites like mitochondria [38], [39], the perinuclear region [40] and in the nuclear membranes of glioblastoma cell lines and biopsy tissues [41], [42]. It is of interest to note that incubation with MNF and CID reduces the total and nuclear expression of MDR proteins and that the reduction of the nuclear expression of Pgp and MRP1 in PANC-1 cells is associated with increased nuclear accumulation of doxorubicin and increased cytotoxicity of doxorubicin and gemcitabine. The MNF-associated increase in cellular and nuclear accumulation of doxorubicin was also observed in MDA-MB-231 breast cancer cells as was the increase in doxorubicin cytotoxicity in MDA-MB-231 and U87MG cancer cells. Both of these cell lines express GPR55 and it is reasonable to assume that the mechanisms associated with the observed effects of MNF and CID on MDR protein expression and function also contribute to increased cytotoxicity in these cell lines.