RUNX2/CBFB modulates the response to MEK inhibitors through activation of receptor tyrosine kinases in KRAS-mutant colorectal cancer

Intrinsic and acquired resistances are major hurdles preventing the effective use of MEK inhibitors for treatment of colorectal cancer (CRC). Some 35–45% of colorectal cancers are KRAS-mutant and their treatment remains challenging as these cancers are refractory to MEK inhibitor treatment, because of feedback activation of receptor tyrosine kinases (RTKs). We reported previously that loss of ERN1 sensitizes a subset of KRAS-mutant colon cancer cells to MEK inhibition. Here we show that the loss of RUNX2 or its cofactor CBFB can confer MEK inhibitor resistance in CRC cells. Mechanistically, we find that cells with genetically ablated RUNX2 or CBFB activate multiple RTKs, which coincides with high SHP2 phosphatase activity, a phosphatase that relays signals from the cell membrane to downstream pathways governing growth and proliferation. Moreover, we show that high activity of SHP2 is causal to loss of RUNX2-induced MEK inhibitor resistance, as a small molecule SHP2 inhibitor reinstates sensitivity to MEK inhibitor in RUNX2 knockout cells. Our results reveal an unexpected role for loss of RUNX2/CBFB in regulating RTK activity in colon cancer, resulting in reduced sensitivity to MEK inhibitors.     

Targeting signal transduction pathways to eliminate chemotherapeutic drug resistance and cancer stem cells

Breast cancer is one of the most common cancers and affects nearly 1 in 7 women. We have demonstrated that targeting the CaM-K, Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be a novel approach to treat drug resistant breast cancer and eliminate cancer stem cells. Common chemotherapeutic drugs, such as doxorubicin, induce the CaM-K pathway which in turn, leads to activation of anti-apoptotic pathways such as Raf/MEK/ERK and PI3K/Akt. Some drug resistant breast cancers exhibited increased expression of CaM-KIV. CaM-K inhibitors synergized with doxorubicin to induce the death of all drug resistant breast cancers examined. Since CaM-Ks are known to result in activation of the Raf/MEK/ERK and PI3K/Akt pathways, we investigated the roles that these pathways exert in breast cancer drug resistance. CaM-K inhibitors suppressed ERK activation in response to doxorubicin in both drug sensitive and resistant cells. CaM-K inhibitors also suppressed ERK activation in response to FBS in the drug resistant cells suggesting dependence on the CaM-K pathway for proliferation. Both the Raf/MEK/ERK and PI3K/Akt pathways are involved in breast cancer drug resistance as they were detected at elevated, activated levels in the drug resistant cells and introduction of constitutively activated forms of Raf-1 and Akt-1 resulted in drug resistance. Drug resistant CICs were often hypersensitive to MEK and mTOR inhibitors, implicating important roles of these pathways in drug resistance. In summary, targeting these pathways may enhance therapy of drug resistant breast cancer and eliminate CICs.Breast cancer therapy is often limited by the occurrence of drug resistance which may be due to the re-emergence of CICs. The studies outlined in this proposal may identify a potentially novel role for CaM-Ks in drug resistance and metastasis and may lead to improved approaches to treat breast tumors by eliminating CICs. Our proposed studies are highly innovative as we will determine the involvement of the CaM-K pathway in breast cancer drug resistance, metastasis and CIC formation. Similar approaches have not been previously performed. Our studies may result in the discovery of novel methods to treat breast cancer by targeting the CaM-K pathway in combination with currently used and approved chemotherapeutic regimens to eliminate CICs which may be responsible for both drug resistance and metastasis.     

Diverse and converging roles of ERK1/2 and ERK5 pathways on mesenchymal to epithelial transition in breast cancer

The epithelial to mesenchymal transition (EMT) is characterized by a loss of cell polarity, a decrease in the epithelial cell marker E-cadherin, and an increase in mesenchymal markers including the zinc-finger E-box binding homeobox (ZEB1). The EMT is also associated with an increase in cell migration and anchorage-independent growth. Induction of a reversal of the EMT, a mesenchymal to epithelial transition (MET), is an emerging strategy being explored to attenuate the metastatic potential of aggressive cancer types, such as triple-negative breast cancers (TNBCs) and tamoxifen-resistant (TAMR) ER-positive breast cancers, which have a mesenchymal phenotype. Patients with these aggressive cancers have poor prognoses, quick relapse, and resistance to most chemotherapeutic drugs. Overexpression of extracellular signal-regulated kinase (ERK) 1/2 and ERK5 is associated with poor patient survival in breast cancer. Moreover, TNBC and tamoxifen resistant cancers are unresponsive to most targeted clinical therapies and there is a dire need for alternative therapies.In the current study, we found that MAPK3, MAPK1, and MAPK7 gene expression correlated with EMT markers and poor overall survival in breast cancer patients using publicly available datasets. The effect of ERK1/2 and ERK5 pathway inhibition on MET was evaluated in MDA-MB-231, BT-549 TNBC cells, and tamoxifen-resistant MCF-7 breast cancer cells. Moreover, TU-BcX-4IC patient-derived primary TNBC cells were included to enhance the translational relevance of our study. We evaluated the effect of pharmacological inhibitors and lentivirus-induced activation or inhibition of the MEK1/2-ERK1/2 and MEK5-ERK5 pathways on cell morphology, E-cadherin, vimentin and ZEB1 expression. Additionally, the effects of pharmacological inhibition of trametinib and XMD8-92 on nuclear localization of ERK1/2 and ERK5, cell migration, proliferation, and spheroid formation were evaluated. Novel compounds that target the MEK1/2 and MEK5 pathways were used in combination with the AKT inhibitor ipatasertib to understand cell-specific responses to kinase inhibition. The results from this study will aid in the design of innovative therapeutic strategies that target cancer metastases.     

Up-regulation of pro-apoptotic protein Bim and down-regulation of anti-apoptotic protein Mcl-1 cooperatively mediate enhanced tumor cell death induced by the combination of ERK kinase (MEK) inhibitor and microtubule inhibitor

Blockade of the ERK signaling pathway by ERK kinase (MEK) inhibitors selectively enhances the induction of apoptosis by microtubule inhibitors in tumor cells in which this pathway is constitutively activated. We examined the mechanism by which such drug combinations induce enhanced cell death by applying time-lapse microscopy to track the fate of individual cells. MEK inhibitors did not affect the first mitosis after drug exposure, but most cells remained arrested in interphase without entering a second mitosis. Low concentrations of microtubule inhibitors induced prolonged mitotic arrest followed by exit of cells from mitosis without division, with most cells remaining viable. However, the combination of a MEK inhibitor and a microtubule inhibitor induced massive cell death during prolonged mitosis. Impairment of spindle assembly checkpoint function by RNAi-mediated depletion of Mad2 or BubR1 markedly suppressed such prolonged mitotic arrest and cell death. The cell death was accompanied by up-regulation of the pro-apoptotic protein Bim (to which MEK inhibitors contributed) and by down-regulation of the anti-apoptotic protein Mcl-1 (to which microtubule and MEK inhibitors contributed synergistically). Whereas RNAi-mediated knockdown of Bim suppressed cell death, stabilization of Mcl-1 by RNAi-mediated depletion of Mule slowed its onset. Depletion of Mcl-1 sensitized tumor cells to MEK inhibitor-induced cell death, an effect that was antagonized by knockdown of Bim. The combination of MEK and microtubule inhibitors thus targets Bim and Mcl-1 in a cooperative manner to induce massive cell death in tumor cells with aberrant ERK pathway activation.     

Dabrafenib; Preclinical Characterization,Increased Efficacy when Combined with Trametinib,while BRAF/MEK Tool Combination Reduced Skin Lesions

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAF^V600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G₁ cell cycle arrest, followed by cell death. In a BRAF^V600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.     

Blockade of the ERK pathway markedly sensitizes tumor cells to HDAC inhibitor-induced cell death

Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway is associated with the neoplastic phenotype of a large number of human tumor cells. Although specific blockade of the ERK pathway by treating such tumor cells with potent mitogen-activated protein kinase/ERK kinase (MEK) inhibitors completely suppresses their proliferation, it by itself shows only a modest effect on the induction of apoptotic cell death. However, these MEK inhibitors markedly enhance the efficacy of histone deacetylase (HDAC) inhibitors to induce apoptotic cell death: such an enhanced cell death is observed only in tumor cells in which the ERK pathway is constitutively activated. Co-administration of MEK inhibitor markedly sensitizes tumor cells to HDAC inhibitor-induced generation of reactive oxygen species, which appears to mediate the enhanced cell death induced by the combination of these agents. These results suggest that the combination of MEK inhibitors and HDAC inhibitors provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway is constitutively activated.     

ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down‐regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.     

Variable apoptotic response of NSCLC cells to inhibition of the MEK/ERK pathway by small molecules or dominant negative mutants

To evaluate the role of the MEK/ERK pathway in NSCLC survival, we analyzed NSCLC cell lines that differed in tumor histology and status of p53, Rb, and K-ras. Constitutive ERK1/2 activity was demonstrated in 17 of 19 cell lines by maintenance of ERK1/2 phosphorylation with serum deprivation. Phosphorylation of ERK1/2 correlated with phosphorylation of MEK1/2 and p90RSK, but was inversely correlated with phosphorylation of c-Raf at S259. With serum deprivation, the MEK inhibitors, PD98059 and U0126, inhibited ERK1/2 activity but did not increase apoptosis. PD98059 and U0126 induced cell cycle arrest in G(0)/G(i) in cells with the highest levels of ERK1/2 activity, which correlated with induction of p27 but not p21. To confirm the cytostatic response to MEK inhibitors, we performed transient transfections with dominant negative forms of MEK or ERK. Surprisingly, dominant negative MEK and ERK mutants increased apoptosis without affecting cell cycle or p27 levels. When combined with paclitaxel, MEK inhibitors had no effect on apoptosis. In contrast, dominant negative ERK2 potentiated paclitaxel-induced apoptosis. Our studies show that constitutive ERK1/2 activity in NSCLC cells promotes cellular survival and chemotherapeutic resistance. Moreover, our data are the first to demonstrate divergent cellular responses to inhibition of the MEK/ERK pathway by small molecule inhibitors or dominant negative mutants.     

Bim protein degradation contributes to cisplatin resistance

Cisplatin is the first-line chemotherapy for the treatment of several cancers. However, the development of cisplatin resistance represents a major clinical problem, and the mechanisms of acquired resistance are not fully understood. Here we show that degradation of the Bcl-2 homology 3-only proapoptotic protein Bim plays an important role in cisplatin resistance in ovarian cancer. Specifically, we show that treatment of ovarian cancer cells with cisplatin caused Bim phosphorylation and subsequent degradation and that its degradation is associated with cisplatin resistance. We also show that cisplatin treatment caused the activation of ERK, which correlated with Bim phosphorylation and degradation. By inhibiting ERK phosphorylation with the MEK inhibitor and knocking down ERK expression with siRNA, we show that Bim phosphorylation and degradation were blocked, which suggests that Bim is phosphorylated by ERK and that such phosphorylation is responsible for cisplatin-induced Bim degradation. We show that ERK was activated in cisplatin-resistant OV433 cells as compared with their counterpart parental OV433 cells. We also show that Bim was phosphorylated and degraded in cisplatin-resistant OV433 cells but not in the parental OV433 cells. Importantly, we show that inhibition of Bim degradation by the proteasome inhibitor MG132 sensitized resistant OV433 cells to cisplatin-induced death. Taken together, our data indicate that degradation of Bim via ERK-mediated phosphorylation can lead to cisplatin resistance. Therefore, these findings suggest that cisplatin resistance can be overcome by the combination of cisplatin and the proteasome inhibitors in ovarian cancer cells.     

Receptor tyrosine kinases in cancer escape from BRAF inhibitors

The BRAF inhibitors (BRAFi) induce anti-tumor responses in nearly 60% of patients with advanced ^V600BRAF-mutant melanomas but only 5% of patients with ^V600BRAF-mutant colorectal carcinomas. Earlier studies of how a subset of melanoma that initially responds to BRAFi but later acquires drug resistance pointed to the importance of receptor tyrosine kinases (RTKs) in drug escape. In a pair of recent reports, this RTK-mediated mechanism of acquired BRAFi resistance in melanoma is re-surfacing in the context of innate or primary BRAFi resistance in ^V600BRAF-mutant colorectal carcinomas, suggesting potential upfront therapeutic strategies to prevent BRAFi resistance.^V600BRAF mutations are found in >50% of melanomas, nearly 100% of hairy cell leukemias but smaller subsets of more common human malignancies (e.g., colorectal, thyroid)¹. The in-human “druggability” of mutant BRAF has been best demonstrated in metastatic BRAF mutant melanomas using the novel small-molecule BRAF inhibitor (BRAFi) PLX4032/vemurafenib, producing survival benefits². Early clinical results of BRAFi in colorectal carcinoma, however, were disappointing, with only 5% of patients (1 of 21 patients) experiencing a partial response and 19% of patients (4 of 21 patients) experiencing minor responses³. This difference in the clinical results (melanoma vs. colorectal carcinoma) may relate less to their ontological origins but more to alternative states of a dynamic and plastic survival signaling network.The majority of BRAF mutant melanomas responds to BRAFi rapidly but acquires drug resistance within a median time of 6-7 months. The specific mechanisms of acquired BRAFi resistance are variegated but fall under two core pathways: 1) reactivation of RAF-MEK-ERK MAPK signaling, and 2) activation of MAPK-redundant signaling via the receptor tyrosine kinase (RTK)-PI3K-AKT pathway, which is parallel but interconnected to the MAPK pathway. MAPK reactivation can occur via NRAS activating mutations⁴, COT overexpression⁵, ^V600BRAF alternative splicing⁶, ^V600BRAF amplification⁷, and MEK1 activating mutation^8,9. MAPK-redundant signaling via RTK overexpression has been shown to result in AKT activation and RAS-CRAF-MEK signaling, bypassing mutant BRAF^4,10,11. The repertoire of RTK overexpressed appears restricted but shares a common pattern of PDGFRβ and EGFR overexpression, at least in melanoma cell lines with acquired resistance to vemurafenib⁴. It is unclear at present how this overexpression of a select number of wild-type RTKs contributes to the molecular details of survival pathway redundancy and cooperativity. Nevertheless, understanding how melanomas acquire BRAFi resistance via core pathways may shed key insights into mechanisms of innate BRAFi resistance in multiple malignancies. Hence, it came as not a complete surprise that a pair of papers published recently implicated RTKs in innate BRAFi resistance in colorectal cancer cell lines^12,13. Both studies pointed to EGFR activation and downstream signaling as a key component to innate BRAFi resistance, at least in a majority of colorectal carcinoma (CRC) cell lines examined.Corcoran et al.¹² showed that BRAF mutant CRC cell lines, in contrast to BRAF mutant melanoma cell lines, displayed innate resistance to growth inhibition by vemurafenib. An important clue implicating RTK involvement in innate vemurafenib resistance of BRAF mutant CRC cell lines came from the observation that p-ERK recovery occurred soon (hours to days) after vemurafenib treatment, unlike the kinetics of p-ERK recovery in BRAF mutant melanoma cell lines. This relatively rapid recovery of p-ERK post vemurafenib treatment in CRC cell lines is akin to that in melanoma cell lines with acquired BRAFi resistance driven by RTK overexpresion¹⁰. Corcoran et al. then traced this propensity for early p-ERK recovery to vemurafenib treatment (24 h)-dependent enhancement of (activated) RAS-GTP levels and MEK activity, parallel to elevated RAS-GTP levels in melanoma cell lines with RTK-driven, acquired BRAFi resistance⁴. In phospho-RTK arrays, they determined that the p-EGFR level (among others such as p-c-MET and p-IGF1R levels) was elevated in CRC cell lines relative to those in melanoma cells. Vemurafenib treatment (24 h) did not significantly enhance the p-EGFR level (but did elevate the p-IGFR1 level). Elevated p-EGFR levels in BRAF mutant CRC cell lines were correlated with elevated total EGFR levels (i.e., overexpressed compared with BRAF mutant melanoma cell lines). Thus, several observations correlated with innate BRAFi resistance in CRC cell lines: RTK (mostly consistently EGFR) overexpression (at baseline); upregulation of activation-associated phosphorylation of RTKs (at baseline); and upregulation of RAS-GTP levels (in response to BRAFi treatment). Curiously, although EGFR is highly phosphorylated at baseline, the RAS-GTP levels only rose in response to vemurafenib treatment.Corcoran et al. further showed that small-molecule EGFR inhibitors (EGFRi) could downregulate, partially or completely, the RAS-GTP level induced by vemurafenib treatment. The combination of vemurafenib (BRAFi) and gefitnib (EGFRi) could synergistically reduce p-ERK levels and the net growth inhibition of most but not all CRC cell lines studied, suggesting that survival in some CRC cell lines may also depend on other RTKs and downstream signaling (e.g., AKT). Consistently, the combination of vemurafenib and erlotinib (EGFRi) stabilized the growth of, but did not cause significant regression of, CRC xenografts. Simultaneous inhibition or genetic knockdown of multiple RTKs was not explored, leaving unresolved the issue of how multiple RTKs may potentially play cooperative survival roles at baseline or in response to kinase inhibitor therapy.Prahallad et al.¹³ also compared CRC and melanoma cell lines and showed that EGFR expression is generally higher in CRC cell lines. Vemurafenib treatment (6 h) of the WiDr CRC cell line led to an induction in p-EGFR and p-AKT levels, concomitant with the expected suppression of p-MEK and p-ERK. MEK inhibition, by AZD6244 treatment, similarly led to the rebound phosphorylation of EGFR. Based on earlier literature showing that the ERK kinase phosphorylates Cdc25c, activating its phosphatase activity, and that Cdc25c can dephosphorylate EGFR, Prahallad et al. went on to show that Cdc25c knockdown mimicked vemurafenib treatment in inducing p-EGFR levels. As predicted, vemurafenib treatment of CRC cell line inhibited Cdc25c phosphorylation at a key threonine (Thr 48), which was previously demonstrated to be a key event for its phosphatase activity. Addition of an EGFRi (cetuximab or gefitnib) to the BRAFi vemurafenib treatment downregulated the baseline level of p-ERK and the BRAFi-induced p-AKT level (but not the baseline p-AKT level). Moreover, addition of an EGFRi sensitized CRC cell lines to growth inhibition by vemurafenib in vitro but did not induce tumor regression in vivo, again suggesting incomplete survival signaling blockade. Accordingly, it has been shown that the effect of vemurafenib in shrinking CRC tumor xenografts was enhanced by combining with an AKT inhibitor (MK-2206)¹⁴. Moreover, in this study, the addition of vemurafenib to erlotinib treatment also resulted in increased anti-tumor activity and improved survival in xenograft models. It should be pointed out that Prahallad et al. did not formally assess BRAFi and EGFRi synergy, nor did they examine the diversity of RTK overexpression/activity and its contribution to downstream survival signaling (e.g., AKT).These works, along with prior studies^4,10, highlight the importance of expression and activity level of RTKs as a key sensitivity determinant of BRAFi resistance in BRAF mutant cancer cell lines (Figure 1). An important question remains as to whether the diversity of RTK overexpression and/or upregulation participates in and contributes to the full BRAFi resistance phenotype. A recent study afforded us a systems-wide view of the RTKinome reprogramming in response to MEK inhibition in the so-called triple-negative breast cancer cell lines¹⁵. The balance of the MAPK vs. RTK network signaling may be dynamically influenced by kinase inhibitors targeting RAF or MEK. This daunting diversity of RTK expression/activity may corner us into abandoning a combination of RTK inhibitors (already approved for clinical usage) with a BRAF inhibitor. Instead, we might need to resort to downstream pathway inhibitors not yet approved for clinical usage (e.g., an inhibitor of MEK with an inhibitor of the PI3K-AKT-mTORC1/2 axis) before we have a chance to corner BRAF mutant cancers into death.Open in a separate windowFigure 1Upregulation of receptor tyrosine kinase(s) (RTKs) as a key sensitivity determinant of BRAFi resistance in BRAF mutant cancer cell lines. (A) In BRAF mutant melanoma cell lines, RTKs are generally expressed at very low levels and contribute minimally to survival signaling, resulting in a strong addiction to mutant BRAF signaling and sensitivity to BRAFi. When BRAF mutant melanoma cell lines acquire BRAFi resistance, they upregulate the expression and activity of PDGFRb and other RTKs, resulting in reactivation of MEK-ERK as well as MAPK-redundant PI3K-AKT survival signaling. (B) In BRAF mutant colorectal carcinoma (CRC) cell lines, EGFR and other RTKs are upregulated by overexpression and some level of activation, resulting in MAPK-redundant survival signaling and conferring innate or primary BRAFi resistance. Treatment of CRC cell lines wth a BRAF or a MEK inhibitor can further activate EGFR and potentially other RTKs and stimulate GTP-RAS levels, consolidating innate BRAFi resistance. Red denotes mutated protein (e.g., BRAF); gray symbols denote weak signaling or interactions; multiplicity of protein symbols denotes overexpression; P in blue denotes activation-associated phosphorylation.     

Evodiamine Synergizes with Doxorubicin in the Treatment of Chemoresistant Human Breast Cancer without Inhibiting P-Glycoprotein

Drug resistance is one of the main hurdles for the successful treatment of breast cancer. The synchronous targeting of apoptosis resistance and survival signal transduction pathways may be a promising approach to overcome drug resistance. In this study, we determined that evodiamine (EVO), a major constituent of the Chinese herbal medicine Evodiae Fructus, could induce apoptosis of doxorubicin (DOX)-sensitive MCF-7 and DOX-resistant MCF-7/ADR cells in a caspase-dependent manner, as confirmed by significant increases of cleaved poly(ADP-ribose) polymerase (PARP), caspase-7/9, and caspase activities. Notably, the reversed phenomenon of apoptosis resistance by EVO might be attributed to its ability to inhibit the Ras/MEK/ERK pathway and the expression of inhibitors of apoptosis (IAPs). Furthermore, our results indicated that EVO enhanced the apoptotic action of DOX by inhibiting the Ras/MEK/ERK cascade and the expression of IAPs without inhibiting the expression and activity of P-glycoprotein (P-gp). Taken together, our data indicate that EVO, a natural product, may be useful applied alone or in combination with DOX for the treatment of resistant breast cancer.     

Vertical inhibition of the MAPK pathway enhances therapeutic responses in NRAS‐mutant melanoma

The MEK inhibitor MEK162 is the first targeted therapy agent with clinical activity in patients whose melanomas harbor NRAS mutations; however, median PFS is 3.7 months, suggesting the rapid onset of resistance in the majority of patients. Here, we show that treatment of NRAS‐mutant melanoma cell lines with the MEK inhibitors AZD6244 or trametinib resulted in a rebound activation of phospho‐ERK (pERK). Functionally, the recovery of signaling was associated with the maintenance of cyclin‐D1 expression and therapeutic escape. The combination of a MEK inhibitor with an ERK inhibitor suppressed the recovery of cyclin‐D1 expression and was associated with a significant enhancement of apoptosis and the abrogation of clonal outgrowth. The MEK/ERK combination strategy induced greater levels of apoptosis compared with dual MEK/CDK4 or MEK/PI3K inhibition across a panel of cell lines. These data provide the rationale for further investigation of vertically co‐targeting the MAPK pathway as a potential treatment option for NRAS‐mutant melanoma patients.     

Tumour cell responses to MEK1/2 inhibitors: acquired resistance and pathway remodelling

The Raf/MEK1/2 [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase 1/2]/ERK1/2 signalling pathway is frequently activated in human tumours due to mutations in BRAF or KRAS. B-Raf and MEK1/2 inhibitors are currently undergoing clinical evaluation, but their ultimate success is likely to be limited by acquired drug resistance. We have used colorectal cancer cell lines harbouring mutations in B-Raf or K-Ras to model acquired resistance to the MEK1/2 inhibitor selumetinib (AZD6244). Selumetinib-resistant cells were refractory to other MEK1/2 inhibitors in cell proliferation assays and exhibited a marked increase in MEK1/2 and ERK1/2 activity and cyclin D1 abundance when assessed in the absence of inhibitor. This was driven by a common mechanism in which resistant cells exhibited an intrachromosomal amplification of their respective driving oncogene, B-Raf V600E or K-RasG13D. Despite the increased signal flux from Raf to MEK1/2, resistant cells maintained in drug actually exhibited the same level of ERK1/2 activity as parental cells, indicating that the pathway is remodelled by feedback controls to reinstate the normal level of ERK1/2 signalling that is required and sufficient to maintain proliferation in these cells. These results provide important new insights into how tumour cells adapt to new therapeutics and highlight the importance of homoeostatic control mechanisms in the Raf/MEK1/2/ERK1/2 signalling cascade.     

ERK pathway positively regulates the expression of Sprouty genes

Sprouty was originally identified as an inhibitor of Drosophila development-associated receptor tyrosine kinase (RTK) signaling. Although RTK signaling has been shown to induce Sprouty gene expression, the precise induction pathway downstream of RTK remains unclear. As RTK signaling pathway includes activation of extracellular signal-regulated kinases (ERKs), we have examined a correlation between activation of ERKs and induction of Sprouty gene expression. All reagents which induce the activation of ERKs induce Sprouty gene expression; these agents include not only growth factors which bind to RTK but also phorbol 12-myristate-13-acetate and active Raf-1 kinase. Furthermore, the Sprouty gene expression induced by all those agents is totally suppressed when the cells are pretreated with specific inhibitors of ERK kinase (MEK). Human tumor cells which exhibit constitutive activation of ERKs show elevated expression of Sprouty genes, which is abolished by treatment of these cells with MEK inhibitors. All these findings clearly indicate that Sprouty gene expression is positively regulated by the ERK pathway downstream of RTK.     

Impact of oncogenic driver mutations on feedback between the PI3K and MEK pathways in cancer cells

Inhibition of the PI3K (phosphoinositide 3-kinase)/Akt/mTORC1 (mammalian target of rapamycin complex 1) and Ras/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathways for cancer therapy has been pursued for over a decade with limited success. Emerging data have indicated that only discrete subsets of cancer patients have favourable responses to these inhibitors. This is due to genetic mutations that confer drug insensitivity and compensatory mechanisms. Therefore understanding of the feedback mechanisms that occur with respect to specific genetic mutations may aid identification of novel biomarkers that predict patient response. In the present paper, we show that feedback between the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways is cell-line-specific and highly dependent on the activating mutation of K-Ras or overexpression c-Met. We found that cell lines exhibited differential signalling and apoptotic responses to PD184352, a specific MEK inhibitor, and PI103, a second-generation class I PI3K inhibitor. We reveal that feedback from the PI3K/Akt/mTORC1 to the Ras/MEK/ERK pathway is present in cancer cells harbouring either K-Ras activating mutations or amplification of c-Met but not the wild-type counterparts. Moreover, we demonstrate that inhibition of protein phosphatase activity by OA (okadaic acid) restored PI103-mediated feedback in wild-type cells. Together, our results demonstrate a novel mechanism for feedback between the PI3K/Akt/mTORC1 and the Ras/MEK/ERK pathways that only occurs in K-Ras mutant and c-Met amplified cells but not the isogenic wild-type cells through a mechanism that may involve inhibition of a specific endogenous phosphatase(s) activity. We conclude that monitoring K-Ras and c-Met status are important biomarkers for determining the efficacy of PI103 and other PI3K/Akt inhibitors in cancer therapy.