Treatment of various MM lines with doses of Dox much lower than LD50 concentrations resulted in phosphorylation of ERK1 and 2, the most abundant ERKs in mammalian cells. In addition to Dox, many other anti-cancer drugs such as paclitaxel and cisplatin induce activation of ERKs in different tumor types [21–23]. However, taxol inhibits ERK activation in different cell types depending upon experimental conditions . In our study, Dox-induced ERK1/2 activation protected MM cells from Dox-induced cell death, as shown when MM lines were pretreated with the MEK1/2 inhibitor, U0126, prior to Dox exposure (Figure 2). In support of our findings, it has been reported that, in most cases, ERK activation protects cells from drug-induced cell death [23, 24], while in some tumor cells, ERK activation contributes to cell death [21, 22]. These different effects may be explained by differences in subcellular distribution of specific ERKs, the longevity of ERK signaling, or phosphorylation of different substrates which may dictate death or survival .
We studied 4 different MM lines for Dox responses after ERK1/2 manipulation either with an inhibitor (U0126) or by shRNA approaches. With the use of the ERK1/2 inhibitor (inhibits both ERK1 and 2), HMESO cells were the best responders (most susceptible) as compared to MO and ME-26 (both lines showed the same susceptibility) (Figure 2A). A shRNA approach to inhibit either ERK1 or ERK2 was studied in 2 MM lines (HMESO and PPMMill). Of the two lines studied by this approach, HMESO again showed more sensitivity to Dox-induced killing after ERK1 or ERK2 inhibition as compared to PPMMill (Figure 2B). In addition, in both cell lines, ERK2 inhibition was more effective than ERK1 inhibition in Dox-induced cell killing (Figure 2B).
Although regulation of apoptotic pathways has been implicated in resistance of many cancers to chemotherapy, we show that human MM lines endogenously overexpress many prosurvival genes (BCL2, cFOS, MET, etc.) in comparison to nontransformed mesothelial cells. The increased levels of these commonly upregulated genes, as reported by our lab and others [18, 26–29] may in part be responsible for drug resistance in MM cell lines. For example, BCL2 and BCL-xL antisense treatment facilitates apoptosis in mesothelioma cells, suggesting BCL2/BCL-xL bispecific antisense treatment in combination with cisplatin or gecitabine may result in a more effective therapy of MM . Consistent with our findings, ERK1/2 activation has been linked to expression and activation of BCL2 in various systems [3, 31] resulting in an anti-apoptotic or survival outcome. cFOS, a protooncogene and component of activator protein-1 (AP-1), is upregulated by crocidolite asbestos in rat pleural mesothelial cells , and endogenously upregulated in human mesothelioma cell lines and tumors [18, 28]. We show for the first time that BRCA1 and BRCA2 are endogenously overexpressed in MM cells, and are pursuing their mutation and functional status in various MMs. ERK1/2 has been linked to feedback regulation of the tumor suppressor/DNA repair gene BRCA1 in irradiation induced DNA damage checkpoint activation [33, 34]. BRCA2 was also endogenously upregulated in MM cells and ERK1/2 inhibition decreased expression of this gene (Table 1), consistent with already published work that ERK1/2 activation inhibits replication of prostate cells via upregulation of BRCA2 . Another gene, PPARγ, which was upregulated only in ME-26 and was significantly inhibited by the U0126 MEK1/2 inhibitor is activated via an ERK1/2 dependent COX-2 pathway in macrophages . Inflammatory pathways involving PPARγ or COX-2 are promising therapeutic targets in a number of cancers . We also report for the first time the upregulation of a cytochrome P450 enzyme gene, CYP3A4, related to drug metabolism in the ME-26 epithelioid cell line that was decreased 3-fold after addition of U0126. The presence of the androgen receptor and its endogenous expression in sarcomatoid MM cells is also a novel finding, and both AR and ESR2 have been linked to the ERK pathway [38–40] as shown in Table 1 in MO cells. A recent study suggests that ER-β affects the prognosis of MM by acting as a tumor suppressor .
ATP-binding cassette (ABC) transporters transport various molecules, including chemotherapeutic drugs, across extra- and intracellular membranes. Increased expression of one or more of these proteins is seen in almost all resistant cancers and is considered responsible fully or in part for the observed drug resistance in most cancer cell lines. In a previous study using MM cell lines, coordinated overexpression of the multi drug resistance pump (MRP) and gamma-glutamylcysteine synthetase genes, but not MDR1, correlated with Dox resistance . In the 3 MM lines we studied by PCR array or microarray analysis, different types of ABC transporter genes were endogenously overexpressed as compared to untransformed LP9/TERT-1 mesothelial cells (Table 1, Table 3). The overexpression of different types of ABC genes in different MM cells further confirms the highly heterogenic nature of MM tumors that vary widely in their prognosis and response to therapy. Inhibition of ABC genes by ERK1 or 2 inhibition may be responsible for the increased accumulation of Dox observed in shERK1 and shERK2 MM cells (Figure 3). Among ABC genes inhibited by shERK2 in HMESO cells, ABCA8 is a relatively uncharacterized new transporter  whereas Dox is a known substrate for ABCC2, ABCA2 and MDR/TAP [44–46]. Our data suggest that different ERKs regulate distinct ABC genes, and a detailed study is needed to understand the roles of different ERKs, including ERK5 that has been linked to chemoresistance in breast cancers , in ABC gene regulation. Consistent with our studies, ERK1 and 2 are linked to regulation of many ABC genes, including ABCG1, ABCA1, MDR1, and MRP1 in various cancer and non-cancer cells [6, 48–50].