CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism

Abstract

Cholangiocarcinoma is a disease with a poor prognosis and increasing incidence and hence there is a pressing unmet clinical need for new adjuvant treatments. Protein kinase CK2 (previously casein kinase II) is a ubiquitously expressed protein kinase that is up-regulated in multiple cancer cell types. The inhibition of CK2 activity using CX-4945 (Silmitasertib) has been proposed as a novel treatment in multiple disease settings including cholangiocarcinoma. Here, we show that CX-4945 inhibited the proliferation of cholangiocarcinoma cell lines in vitro. Moreover, CX-4945 treatment induced the formation of cytosolic vacuoles in cholangiocarcinoma cell lines and other cancer cell lines. The vacuoles contained extracellular fluid and had neutral pH, features characteristic of methuosis. In contrast, simultaneous knockdown of both the α and α′ catalytic subunits of protein kinase CK2 using small interfering RNA (siRNA) had little or no effect on the proliferation of cholangiocarcinoma cell lines and failed to induce the vacuole formation. Surprisingly, low doses of CX-4945 increased the invasive properties of cholangiocarcinoma cells due to an upregulation of matrix metallopeptidase 7 (MMP-7), while the knockdown of CK2 inhibited cell invasion. Our data suggest that CX-4945 inhibits cell proliferation and induces cell death via CK2-independent pathways. Moreover, the increase in cell invasion brought about by CX-4945 treatment suggests that this drug might increase tumor invasion in clinical settings.

Role of CK2 inhibitor CX-4945 in anti-cancer combination therapy – potential clinical relevance

“…We and others have previously reported that the induction of methuosis mediated by CX-4945, as well as its derivative CX-5011, is CK2-independent [93,95]. Experimental data exonerate CK2 from being involved in the activation of macropinocytosis, since both the genetic reduction/ablation of CK2 subunits and the treatment of cells with kinase inhibitors structurally unrelated to CX-4945, failed to promote the aberrant formation of the cytosolic vesicles [93,95]. Moreover, we showed that the ability of CX-4945 to promote methuotic cell death was cell type-specific since the extent of the vacuolization that followed CX-4945 stimulation considerably differed amongst the various cell lines used, distinguishing highly sensitive cells (i.e., HepG2 and GN11), moderately sensitive cells (i.e., MDA-MB-231 and HEK-293 T) and insensitive cells (i.e., HeLa).…”

“…Finally, it is to be mentioned that CX-4945 has recently been characterized as a methuosis inducer, a new issue that could be relevant in anti-cancer combination therapy. When used at high micromolar concentrations, ranging from 10 to 50 μM, CX-4945 promotes a distinctive form of cell death characterized by displacement of large, macro-pinocytic-derived and fluid-filled cytosolic vacuoles, that has been dubbed methuosis (from the Greek word μεθύω, to be drunk) [93][94][95]. We and others have previously reported that the induction of methuosis mediated by CX-4945, as well as its derivative CX-5011, is CK2-independent [93,95].…”

“…When used at high micromolar concentrations, ranging from 10 to 50 μM, CX-4945 promotes a distinctive form of cell death characterized by displacement of large, macro-pinocytic-derived and fluid-filled cytosolic vacuoles, that has been dubbed methuosis (from the Greek word μεθύω, to be drunk) [93][94][95]. We and others have previously reported that the induction of methuosis mediated by CX-4945, as well as its derivative CX-5011, is CK2-independent [93,95]. Experimental data exonerate CK2 from being involved in the activation of macropinocytosis, since both the genetic reduction/ablation of CK2 subunits and the treatment of cells with kinase inhibitors structurally unrelated to CX-4945, failed to promote the aberrant formation of the cytosolic vesicles [93,95].…”

Epimedokoreanin C, A Prenylated Flavonoid Isolated from Epimedium Koreanum, Induces Non-Apoptotic Cell Death with the Characteristics of Methuosis in Lung Cancer Cells

“…As a unique form of non-apoptotic cell death, methuosis was initially de ned in glioblastoma cells after overexpression of Ras [33]. In recent years, some methuosis inducers were identi ed and all of these compounds displayed meaningful effects in killing cancer cells [10][11][12][13][14][15]. This indicated that small molecules with the capacity to induce methuosis may be of considerable interest as potential therapeutics for cancers that are resistant to apoptosis.…”

“…As a novel nonapoptotic mode of cell death, methuosis has attracted particular attention as a target for cancer treatments. Several methuosis inducers have been reported, for example, indole-based chalcones MIPP and MOMIPP triggered cell death by methuosis in glioblastoma [10,11]; an ursolic acid derivative led cell death via methuosis [12]; a 4,6-disubstituted Aza-indole compound 13 induced methuosis and displayed cytotoxicities against a panel of cancer cells [13]; a quinoline derivative vacquinol also caused methuosis in glioblastoma [14] and CX-4945 induced methuosis in cholangiocarcinoma cells [15]. These reported results con rmed that the identi cation of methuosis inducers could provide both probes for studying the speci c molecular mechanism of the nonconventional cell death pathway, and promising therapeutic agents for the treatment of cancers.…”