Connexin 43 Inhibition Sensitizes Chemoresistant Glioblastoma Cells to Temozolomide

Abstract

Resistance of glioblastoma (GBM) to the front-line chemotherapeutic agent temozolomide (TMZ) continues to challenge GBM treatment efforts. The repair of TMZ-induced DNA damage by O-6-methylguanine-DNA methyltransferase (MGMT) confers one mechanism of TMZ resistance. Paradoxically, MGMT-deficient GBM patients survive longer despite still developing resistance to TMZ. Recent studies indicate that the gap junction protein connexin 43 (Cx43) renders GBM cells resistant to TMZ through its carboxyl terminus (CT). In this study, we report insights into how Cx43 promotes TMZ resistance. Cx43 levels were inversely correlated with TMZ sensitivity of GBM cells, including GBM stem cells. Moreover, Cx43 levels inversely correlated with patient survival, including as observed in MGMT-deficient GBM patients. Addition of the C-terminal peptide mimetic αCT1, a selective inhibitor of Cx43 channels, sensitized human MGMT-deficient and TMZ-resistant GBM cells to TMZ treatment. Moreover, combining αCT1 with TMZ blocked AKT/mTOR signaling, induced autophagy and apoptosis in TMZ-resistant GBM cells. Our findings suggest that Cx43 may offer a biomarker to predict the survival of patients with MGMT-independent TMZ resistance, and that combining a Cx43 inhibitor with TMZ could enhance therapeutic responses in GBM and perhaps other TMZ-resistant cancers.

Novel approach to temozolomide resistance in malignant glioma: connexin43-directed therapeutics

“…For example, dibutyryl cyclic adenosine monophosphate was found to sensitize medulloblastoma cells to TMZ, possibly through increasing the expression of Cx43 and activating a Cx43-induced bystander effect [23]. We recently found that Cx43 levels were notably high in 2 out of 9 GBM cell lines and 4 out of 6 primary GBM samples [6 •• ]. This variability in Cx43 expression levels suggests that this GJ protein plays divergent roles in GBMs.…”

“…MGMT effectively repairs TMZ-induced DNA damage, thereby limiting TMZ efficacy in GBM patients expressing this gene. However, GBM patients with no or limited expression of MGMT also develop TMZ resistance [5,6 •• ]. Interestingly, in contrast to growth inhibition induced by a reinforced expression of Cx43 in C6 malignant glioma cells [18], several studies have found that ectopically expressing Cx43 in GBM cells confers TMZ resistance.…”

“…Lai and colleagues exposed Cx43-low/TMZ-sensitive U251 GBM cells to a low dose of TMZ to select a TMZ-resistant U251 clone and subsequently found that TMZ-resistant U251 cells expressed higher levels of Cx43 and, intriguingly, that the MGMT gene showed reactivated expression in the TMZ-resistant clone [45 • ]. To clarify whether endogenous Cx43 confers TMZ resistance in GBM, we measured levels of Cx43 in 7 MGMT-deficient GBM cell lines and found that lower levels of Cx43 strongly correlated with the increased sensitivity of GBM cells to TMZ when MGMT was not present, suggesting that high levels of Cx43 contribute to MGMT-independent TMZ resistance [6 •• ]. In addition, a GSC line LN229/GSC derived from LN229 GBM cells, exhibited a lower sensitivity to TMZ coinciding with an elevated level of Cx43.…”

“…As a result, tumor cells expressing high levels of MGMT display significant resistance to TMZ therapy [4]. However, sensitivity of GBM cells to TMZ may also be independent of MGMT expression [5,6 •• ], highlighting the challenges presented by cellular heterogeneity in malignant glioma and the need for novel approaches in eliminating such refractory tumor cell populations to successfully treat this disease and prevent recurrence.…”

“…An added layer of complexity is afforded through channel-independent mechanisms via the stabilization or destabilization of multiprotein complexes mediated through protein binding sites on the connexin protein. Connexin43 (Cx43) is the most ubiquitous connexin and in addition to having critical regulatory and developmental roles in many tissues, has been implicated as a driver of tumor invasion [11 •• ], a marker of tumor progression [12], and an inducer of TMZ resistance in GBM cells [6 •• ,13–15,16 •• ]. Furthermore, the expression and function of connexins in the microenvironment of malignant glioma is an evolving field, where recent studies investigating the function of Cx43 in the tumor microenvironment reveal that Cx43 in peritumoral cells regulates gliomagenesis, tumor progression, and treatment resistance.…”