PI3K/Akt-sensitive MEK-independent compensatory circuit of ERK activation in ER-positive PI3K-mutant T47D breast cancer cells

We explored the crosstalk between cell survival (phosphatidylinositol 3-kinase (PI3K)/Akt) and mitogenic (Ras/Raf/MEK/extracellular signal-regulated kinase (ERK)) signaling pathways activated by an epidermal growth factor (EGF) and analyzed their sensitivity to small molecule inhibitors in the PI3K-mutant estrogen receptor (ER)-positive MCF7 and T47D breast cancer cells. In contrast to MCF7 cells, ERK phosphorylation in T47D cells displayed resistance to MEK inhibition by several structurally different compounds, such as U0126, PD 098059 and PD 198306, MEK suppression by small interfering RNA (siRNA) and was also less sensitive to PI3K inhibition by wortmannin. Similar effect was observed in PI3K-wild type ER-positive BT-474 cells, albeit to a much lesser extent.MEK-independent ERK activation was induced only by ErbB receptor ligands and was resistant to inhibition of several kinases and phosphatases that are known to participate in the regulation of Ras/mitogen-activated protein kinase (MAPK) cascade. Although single agents against PDK1 or Akt did not affect EGF-induced ERK phosphorylation, a combination of PI3K/Akt and MEK inhibitors synergistically suppressed ERK activation and cellular growth. siRNA-mediated silencing of class I PI3K or Akt1/2 genes also significantly decreased U0126-resistant ERK phosphorylation.Our data suggest that in T47D cells ErbB family ligands induce a dynamic, PI3K/Akt-sensitive and MEK-independent compensatory ERK activation circuit that is absent in MCF7 cells. We discuss candidate proteins that can be involved in this activation circuitry and suggest that PDZ-Binding Kinase/T-LAK Cell-Originated Protein Kinase (PBK/TOPK) may play a role in mediating MEK-independent ERK activation.     

eEF2K Activity Determines Synergy to Cotreatment of Cancer Cells With PI3K and MEK Inhibitors

PI3K-mammalian target of rapamycin and MAPK/ERK kinase (MEK)/mitogen-activated protein kinase (MAPK) are the most frequently dysregulated signaling pathways in cancer. A problem that limits the success of therapies that target individual PI3K-MAPK members is that these pathways converge to regulate downstream functions and often compensate each other, leading to drug resistance and transient responses to therapy. In order to overcome resistance, therapies based on cotreatments with PI3K/AKT and MEK/MAPK inhibitors are now being investigated in clinical trials, but the mechanisms of sensitivity to cotreatment are not fully understood. Using LC-MS/MS-based phosphoproteomics, we found that eukaryotic elongation factor 2 kinase (eEF2K), a key convergence point downstream of MAPK and PI3K pathways, mediates synergism to cotreatment with trametinib plus pictilisib (which target MEK1/2 and PI3Kα/δ, respectively). Inhibition of eEF2K by siRNA or with a small molecule inhibitor reversed the antiproliferative effects of the cotreatment with PI3K plus MEK inhibitors in a cell model–specific manner. Systematic analysis in 12 acute myeloid leukemia cell lines revealed that eEF2K activity was increased in cells for which PI3K plus MEKi cotreatment is synergistic, while PKC potentially mediated resistance to such cotreatment. Together, our study uncovers eEF2K activity as a key mediator of responses to PI3Ki plus MEKi and as a potential biomarker to predict synergy to cotreatment in cancer cells.     

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.     

PBK/TOPK在乳腺癌细胞中介导MEK非依赖性ERK激活中作用机制分析

该文探讨了乳腺癌细胞中表皮生长因子(EGF)介导的MEK非依赖性ERK激活通路。Western blot检测EGF刺激下,siRNA抑制MEK1/2后的T47D细胞的p-ERK水平,以验证T47D细胞中存在EGF介导的MEK非依赖性ERK激活的通路。接着使用可能参与MEK非依赖性ERK激活的激酶的小分子抑制剂抑制相关激酶(AC、PKC、Src、PI3K、PDK1和Akt)活性后,检测T47D细胞EGF介导ERK的磷酸化水平。siRNA抑制MEK1/2表达后,T47D细胞在EGF刺激后的仍保留部分p-ERK,即在T47D细胞中,存在EGF介导的MEK非依赖性的ERK磷酸化通路。小分子抑制剂抑制AC、PKC、Src对MEK非依赖性ERK激活途径影响不大。而使用小分子抑制剂抑制PI3K、PDK1和Akt后,ERK的磷酸化水平显著降低,提示PI3K/Akt通路下游的激酶参与T47D中EGF介导的MEK非依赖性ERK激活途径。siRNA干扰PI3K/Akt通路下游PBK/TOPK后并使用U0126抑制MEK功能后,几乎检测不到p-ERK,提示PBK/TOPK参与T47D细胞中EGF介导的MEK非依赖性ERK激活途径。乳腺癌抗雌激素药物耐药株T47D细胞存在EGF介导的MEK非依赖性ERK激活途径,且该途径受PI3K/Akt下游的PBK/TOPK调控。     

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.     

sst2 Somatostatin receptor inhibits cell proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation

The G protein-coupled sst2 somatostatin receptor is a critical negative regulator of cell proliferation. sstII prevents growth factor-induced cell proliferation through activation of the tyrosine phosphatase SHP-1 leading to induction of the cyclin-dependent kinase inhibitor p27Kip1. Here, we investigate the signaling molecules linking sst2 to p27Kip1. In Chinese hamster ovary-DG-44 cells stably expressing sst2 (CHO/sst2), the somatostatin analogue RC-160 transiently stimulates ERK2 activity and potentiates insulin-stimulated ERK2 activity. RC-160 also stimulates ERK2 activity in pancreatic acini isolated from normal mice, which endogenously express sst2, but has no effect in pancreatic acini derived from sst2 knock-out mice. RC-160-induced p27Kip1 up-regulation and inhibition of insulin-dependent cell proliferation are both prevented by pretreatment of CHO/sst2 cells with the MEK1/2 inhibitor PD98059. In addition, using dominant negative mutants, we show that sst2-mediated ERK2 stimulation is dependent on the pertussis toxin-sensitive Gi/o protein, the tyrosine kinase Src, both small G proteins Ras and Rap1, and the MEK kinase B-Raf but is independent of Raf-1. Phosphatidylinositol 3-kinase (PI3K) and both tyrosine phosphatases, SHP-1 and SHP-2, are required upstream of Ras and Rap1. Taken together, our results identify a novel mechanism whereby a Gi/o protein-coupled receptor inhibits cell proliferation by stimulating ERK signaling via a SHP-1-SHP-2-PI3K/Ras-Rap1/B-Raf/MEK pathway.     

Involvement of Akt and mTOR in chemotherapeutic- and hormonal-based drug resistance and response to radiation in breast cancer cells

Elucidating the response of breast cancer cells to chemotherapeutic and hormonal based drugs and radiation is clearly important as these are common treatment approaches. Signaling cascades often involved in chemo-, hormonal- and radiation resistance are the Ras/PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK and p53 pathways. In the following studies we have examined the effects of activation of the Ras/PI3K/PTEN/Akt/mTOR cascade in the response of MCF-7 breast cancer cells to chemotherapeutic- and hormonal-based drugs and radiation. Activation of Akt by introduction of conditionally-activated Akt-1 gene could result in resistance to chemotherapeutic and hormonal based drugs as well as radiation. We have determined that chemotherapeutic drugs such as doxorubicin or the hormone based drug tamoxifen, both used to treat breast cancer, resulted in the activation of the Raf/MEK/ERK pathway which is often associated with a pro-proliferative, anti-apoptotic response. In drug sensitive MCF-7 cells which have wild-type p53; ERK, p53 and downstream p21^Cip-1 were induced upon exposure to doxorubicin. In contrast, in the drug resistant cells which expressed activated Akt-1, much lower levels of p53 and p21^Cip1 were induced upon exposure to doxorubicin. These results indicate the involvement of the Ras/PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK and p53 pathways in the response to chemotherapeutic and hormonal based drugs. Understanding how breast cancers respond to chemo- and hormonal-based therapies and radiation may enhance the ability to treat breast cancer more effectively.     

Synergistic Effects of Concurrent Blockade of PI3K and MEK Pathways in Pancreatic Cancer Preclinical Models

Patients with pancreatic cancer have dismal prognoses, and novel therapies are urgently needed. Mutations of the KRAS oncogene occur frequently in pancreatic cancer and represent an attractive target. Direct targeting of the predominant KRAS pathways have been challenging and research into therapeutic strategies have been now refocused on pathways downstream of KRAS, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK [MEK]). We hypothesized that concurrent inhibition of the PI3K and MEK pathways would result in synergistic antitumor activity, as it would circumvent the compensatory feedback loop between the two pathways. We investigated the combined effect of the PI3K inhibitor, GDC0941, and the MEK inhibitor, AZD6244, on cell viability, apoptosis and cell signaling in a panel of pancreatic cancer cell lines. An in vivo analysis was conducted on pancreatic cancer xenografts. While BxPC-3 (KRAS wild type) and MIA PaCa-2 (KRAS mutated) cell lines were sensitive to GDC0941 and AZD6244 as single agents, synergistic inhibition of tumor cell growth and induction of apoptosis were observed in both cell lines when the two drugs were combined. Interestingly, phosphorylation of the cap-dependent translational components, 4E-binding protein (p-4E-BP1) and S6 was found to be closely associated with sensitivity to GDC0941 and AZD6244. In BxPC-3 cell xenografts, survival differences were observed between the control and the AZD6244, GDC0941, and combination groups. Our study provides the rationale for concurrent targeting of the PI3K and MEK pathways, regardless of KRAS status, and suggests that phosphorylation of 4E-BP1and S6 can serve as a predictive biomarker for response to treatment.     

Involvement of Akt and mTOR in chemotherapeutic and hormonal-based drug resistance and response to radiation in breast cancer cells

Elucidating the response of breast cancer cells to chemotherapeutic and hormonal based drugs and radiation is clearly important as these are common treatment approaches. Signaling cascades often involved in chemo-, hormonal- and radiation resistance are the Ras/PI3K/PTE N/Akt/mTO R, Ras/Raf/MEK/ERK and p53 pathways. In the following studies we have examined the effects of activation of the Ras/PI3K/PTE N/Akt/mTO R cascade in the response of MCF-7 breast cancer cells to chemotherapeutic- and hormonal-based drugs and radiation. Activation of Akt by introduction of conditionally-activated Akt-1 gene could result in resistance to chemotherapeutic and hormonal based drugs as well as radiation. We have determined that chemotherapeutic drugs such as doxorubicin or the hormone based drug tamoxifen, both used to treat breast cancer, resulted in the activation of the Raf/MEK/ERK pathway which is often associated with a proproliferative, anti-apoptotic response. In drug sensitive MCF-7 cells which have wild-type p53; ERK, p53 and downstream p21^Cip-1 were induced upon exposure to doxorubicin. In contrast, in the drug resistant cells which expressed activated Akt-1, much lower levels of p53 and p21^Cip1 were induced upon exposure to doxorubicin. These results indicate the involvement of the Ras/PI3K/PTE N/Akt/mTO R, Ras/Raf/MEK/ERK and p53 pathways in the response to chemotherapeutic and hormonal based drugs. Understanding how breast cancers respond to chemo- and hormonal-based therapies and radiation may enhance the ability to treat breast cancer more effectively.Key words: Akt, ERK, mTOR, chemotherapeutic drugs, radiation     

K-RAS Mutant Pancreatic Tumors Show Higher Sensitivity to MEK than to PI3K Inhibition In Vivo

Activating K-RAS mutations occur at a frequency of 90% in pancreatic cancer, and to date no therapies exist targeting this oncogene. K-RAS signals via downstream effector pathways such as the MAPK and the PI3K signaling pathways, and much effort has been focused on developing drugs targeting components of these pathways. To better understand the requirements for K-RAS and its downstream signaling pathways MAPK and PI3K in pancreatic tumor maintenance, we established an inducible K-RAS knock down system that allowed us to ablate K-RAS in established tumors. Knock down of K-RAS resulted in impaired tumor growth in all pancreatic xenograft models tested, demonstrating that K-RAS expression is indeed required for tumor maintenance of K-RAS mutant pancreatic tumors. We further examined signaling downstream of K-RAS, and detected a robust reduction of pERK levels upon K-RAS knock down. In contrast, no effect on pAKT levels could be observed due to almost undetectable basal expression levels. To investigate the requirement of the MAPK and the PI3K pathways on tumor maintenance, three selected pancreatic xenograft models were tested for their response to MEK or PI3K inhibition. Tumors of all three models regressed upon MEK inhibition, but showed less pronounced response to PI3K inhibition. The effect of MEK inhibition on pancreatic xenografts could be enhanced further by combined application of a PI3K inhibitor. These data provide further rationale for testing combinations of MEK and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring mutations in K-RAS.     

Pharmacological inhibition of the MEK5/ERK5 and PI3K/Akt signaling pathways synergistically reduces viability in triple-negative breast cancer

Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.     

PAK4 confers cisplatin resistance in gastric cancer cells via PI3K/Akt- and MEK/ERK-dependent pathways

CDDP [cisplatin or cis-diamminedichloroplatinum(II)] and CDDP-based combination chemotherapy have been confirmed effective against gastric cancer. However, CDDP efficiency is limited because of development of drug resistance. In this study, we found that PAK4 (p21-activated kinase 4) expression and activity were elevated in gastric cancer cells with acquired CDDP resistance (AGS/CDDP and MKN-45/CDDP) compared with their parental cells. Inhibition of PAK4 or knockdown of PAK4 expression by specific siRNA (small interfering RNA)-sensitized CDDP-resistant cells to CDDP and overcome CDDP resistance. Combination treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 [the inhibitor of PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B or PKB) pathway] or PD98509 {the inhibitor of MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] pathway} with PF-3758309 (the PAK4 inhibitor) resulted in increased CDDP efficacy compared with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or PD98509 alone. However, after the concomitant treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PD98509, PF-3758309 administration exerted no additional enhancement of CDDP cytotoxicity in CDDP-resistant cells. Inhibition of PAK4 by PF-3758309 could significantly suppress MEK/ERK and PI3K/Akt signalling in CDDP-resistant cells. Furthermore, inhibition of PI3K/Akt pathway while not MEK/ERK pathway could inhibit PAK4 activity in these cells. The in vivo results were similar with those of in vitro. In conclusion, these results indicate that PAK4 confers CDDP resistance via the activation of MEK/ERK and PI3K/Akt pathways. PAK4 and PI3K/Akt pathways can reciprocally activate each other. Therefore, PAK4 may be a potential target for overcoming CDDP resistance in gastric cancer.     

Dual Inhibition of PI3K/AKT and MEK/ERK Pathways Induces Synergistic Antitumor Effects in Diffuse Intrinsic Pontine Glioma Cells

Diffuse intrinsic pontine glioma (DIPG) is a devastating disease with an extremely poor prognosis. Recent studies have shown that platelet-derived growth factor receptor (PDGFR) and its downstream effector pathway, PI3K/AKT/mTOR, are frequently amplified in DIPG, and potential therapies targeting this pathway have emerged. However, the addition of targeted single agents has not been found to improve clinical outcomes in DIPG, and targeting this pathway alone has produced insufficient clinical responses in multiple malignancies investigated, including lung, endometrial, and bladder cancers. Acquired resistance also seems inevitable. Activation of the Ras/Raf/MEK/ERK pathway, which shares many nodes of cross talk with the PI3K/AKT pathway, has been implicated in the development of resistance. In the present study, perifosine, a PI3K/AKT pathway inhibitor, and trametinib, a MEK inhibitor, were combined, and their therapeutic efficacy on DIPG cells was assessed. Growth delay assays were performed with each drug individually or in combination. Here, we show that dual inhibition of PI3K/AKT and MEK/ERK pathways synergistically reduced cell viability. We also reveal that trametinib induced AKT phosphorylation in DIPG cells that could not be effectively attenuated by the addition of perifosine, likely due to the activation of other compensatory mechanisms. The synergistic reduction in cell viability was through the pronounced induction of apoptosis, with some effect from cell cycle arrest. We conclude that the concurrent inhibition of the PI3K/AKT and MEK/ERK pathways may be a potential therapeutic strategy for DIPG.     

KRASG12D- and BRAFV600E-Induced Transformation of Murine Pancreatic Epithelial Cells Requires MEK/ERK-Stimulated IGF1R Signaling

Mutation of KRAS is a common initiating event in pancreatic ductal adenocarcinoma (PDAC). Yet, the specific roles of KRAS-stimulated signaling pathways in the transformation of pancreatic ductal epithelial cells (PDEC), putative cells of origin for PDAC, remain unclear. Here, we show that KRAS(G12D) and BRAF(V600E) enhance PDEC proliferation and increase survival after exposure to apoptotic stimuli in a manner dependent on MEK/ERK and PI3K/AKT signaling. Interestingly, we find that activation of PI3K/AKT signaling occurs downstream of MAP-ERK kinase (MEK), and is dependent on the autocrine activation of the insulin-like growth factor (IGF) receptor (IGF1R) by IGF2. Importantly, IGF1R inhibition impairs KRAS(G12D)- and BRAF(V600E)-induced survival, whereas ectopic IGF2 expression rescues KRAS(G12D)- and BRAF(V600E)-mediated survival downstream of MEK inhibition. Moreover, we show that KRAS(G12D)- and BRAF(V600E)-induced tumor formation in an orthotopic model requires IGF1R. Interestingly, we show that while individual inhibition of MEK or IGF1R does not sensitize PDAC cells to apoptosis, their concomitant inhibition reduces survival. Our findings identify a novel mechanism of PI3K/AKT activation downstream of activated KRAS, illustrate the importance of MEK/ERK, PI3K/AKT, and IGF1R signaling in pancreatic tumor initiation, and suggest potential therapeutic strategies for this malignancy. Mol Cancer Res; 10(9); 1228-39. ?2012 AACR.     

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.     

A novel SND1-BRAF fusion confers resistance to c-Met inhibitor PF-04217903 in GTL16 cells though MAPK activation

Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling.     

Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells

Docosahexaenoic acid (22: 6n-3; DHA) is a long chain polyunsaturated fatty acid that exists highly enriched in fish oil, and it is one of the low molecular weight food chemicals which can pass a blood brain barrier. A preliminary survey of several fatty acids for expression of growth-associated protein-43 (GAP-43), a marker of axonal growth, identified DHA as one of the most potent inducers. The human neuroblastoma SH-SY5Y cells exposed to DHA showed significant and dose-dependent increases in the percentage of cells with longer neurites. To elucidate signaling mechanisms involved in DHA-enhanced basal neuritogenesis, we examined the role of extracellular signal-regulated kinase (ERK)1/2 and intracellular reactive oxygen species (ROS) production using SH-SY5Y cells. From immunoblotting experiments, we observed that DHA induced the ROS production, protein tyrosine phosphatase inhibition, mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) phosphorylation, and sequentially ERK1/2 phosphorylation, the last of which was significantly reduced by MEK inhibitor U0126. Both antioxidants and MEK inhibitor affected DHA-induced GAP-43 expression, whereas the specific PI3K inhibitor LY294002 did not. We found that total protein tyrosine phosphatase activity was also downregulated by DHA treatment, which was counteracted by antioxidant pretreatment. These results suggest that the ROS-dependent ERK pathway, rather than PI3K, plays an important role during DHA-enhanced neurite outgrowth.