Protein kinase C alpha trigger Ras and Raf-independent MEK/ERK activation for TPA-induced growth inhibition of human hepatoma cell HepG2

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a well-known activator of both protein kinase C (PKC) and mitogen activated protein kinase (MAPK) signal cascade triggering a lot of effects in many non-tumor and tumor cells. We have reported activation of PKCalpha isozyme was specifically required for TPA-induced ERK (MAPK) signaling that mediated gene expressions of the CDK inhibitors p15(INK4b) and p16 (INK4a) leading to growth inhibition of hepatoma cell HepG2. We further investigated the upstream signal molecule linking PKCalpha to ERK. In the Ras activation assay, HepG2 cell exhibited substantial amount of Ras activity. Treatment of the cell with 50nM TPA for 10min slightly inhibited Ras activity by about 10-20%. Pretreatment of the cell with 10microM manumycin A, which abolish basal Ras activity, did not prevent TPA-triggered ERK phosphorylation. Immunoprecipitation coupled with kinase assay demonstrated that MEK-1 activity was strongly induced by treatment of TPA for 5-30min in HepG2. In contrast, c-Raf activity was not significantly induced by TPA within 5-15min. Consistently, Western blot of Phospho(ser-218/222)-MEK demonstrated that phosphorylation of MEK-1 was greatly induced by 50nM TPA, which can be prevented by the PKC inhibitor Bisindolylmaleimides II. Moreover, pretreatment of the MEK1/2 inhibitor, but not c-Raf inhibitor prevented the TPA-induced ERK phosphorylation, gene expression of p15(INK4b) and p16 (INK4a) and growth inhibition of HepG2. In addition, transient expression of a dominant negative Raf mutant in HepG2 did not prevent these effects of TPA. Constitutive expression of an active PKCalpha mutant in HepG2 enhanced phosphorylation of both MEK and ERK accompanied with induction of gene expression of p16(INK4a) and growth inhibition of HepG2. In contrast, Ras and Raf activity were not increased by expression of active PKCalpha. Taken together, we conclude that PKCalpha may activate MEK, independently of Raf and Ras, to trigger sustained ERK (MAPK) signaling and cell cycle arrest of HepG2 induced by TPA.     

Radiosensitization and downregulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) upon inhibition of mitogen/extracellular signal-regulated kinase (MEK) in malignant melanoma cells

Background

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an important cofactor in the p53-mediated DNA damage response pathway upon ionizing radiation (IR) and exerts anti-apoptotic effects also independent of p53 pathway activation. Furthermore, hnRNP K is overexpressed in various neoplasms including malignant melanoma (MM). Here, we investigate the role of hnRNP K in the radioresistance of MM cells.

Methods and results

Our results show cytoplasmic expression of hnRNP K in human MM surgical specimens, but not in benign nevi, and a quick dose- and time-dependent upregulation in response to IR accompanied by cytoplasmic redistribution of the protein in the IPC-298 cellular tumor model carrying an activating NRAS mutation (p.Q61L). SiRNA-based knockdown of hnRNP K induced a delayed decline in γH2AX/53BP1-positive DNA repair foci upon IR. Pharmacological interference with MAPK signaling abrogated ERK phosphorylation, diminished cellular hnRNP K levels, impaired γH2AX/53BP1-foci repair and proliferative capability and increased apoptosis comparable to the observed hnRNP K knockdown phenotype in IPC-298 cells.

Conclusion

Our results indicate that pharmacological interference with MAPK signaling increases vulnerability of NRAS-mutant malignant melanoma cells to ionizing radiation along with downregulation of endogenous hnRNP K and point towards a possible use for combined MEK inhibition and localized radiation therapy of MM in the NRAS-mutant setting where BRAF inhibitors offer no clinical benefit.     

Roles of the PI-3K and MEK pathways in Ras-mediated chemoresistance in breast cancer cells

Activated Ras utilises several downstream pathways, including the mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway and the phosphoinositide 3-kinase (PI-3k)/Akt pathway, to promote cell proliferation and to inhibit apoptosis. To investigate which pathway plays a major role in Ras-induced drug resistance to chemotherapeutic agents in breast cancer cells, we transfected MCF7 breast cancer cells with a constitutively active H-RasG12V and examined the toxicities of three commonly used breast cancer chemotherapeutic agents, paclitaxel, doxorubicin, and 5-fluorouracil in these cells under the conditions that PI-3K or MEK were selectively inhibited by their respective specific inhibitors or dominant negative expression vectors. We found that Ras-mediated drug resistance is well correlated with resistance to apoptosis induced by anticancer agents in MCF7 breast cancer cells. Although inhibition of MEK/MAPK or PI-3K/Akt can each enhance the cytotoxicity of paclitaxel, doxorubicin, or 5-fluorouracil, inhibition of the PI-3K/Akt pathway seems to have a greater effect than inhibition of the MEK/MAPK pathway in reversing Ras-mediated drug resistance. Our results indicate that the PI-3K pathway may play a more important role in receptor tyrosine kinase-mediated resistance to chemotherapy and suggest that PI-3K/Akt might be a critical target molecule for anticancer intervention in breast cancer.     

Regulation of cell cycle progression and apoptosis by the Ras/Raf/MEK/ERK pathway (Review)

The Ras/Raf/MEK/ERK signal transduction pathway regulates cell cycle progression and apoptosis in diverse types of cells. Mutations in this pathway are often observed in transformed cell lines and frequently linked with human cancers. The Ras/Raf/MEK/ERK pathway can induce events both associated with cell proliferation and cell cycle arrest. The particular course chosen may depend on the strength and the particular Raf gene activated by Ras. This pathway also is involved in maintaining cell survival by modulating the activity of apoptotic molecules including Bad and Bcl-2. This review will discuss the regulation of the Ras/Raf/MEK/ERK pathway and how it modulates cell cycle progression and cell survival.     

The TGF-beta co-receptor endoglin modulates the expression and transforming potential of H-Ras

Endoglin is a coreceptor for transforming growth factor-β (TGF-β) that acts as a suppressor of malignancy during mouse skin carcinogenesis. Because in this model system H-Ras activation drives tumor initiation and progression, we have assessed the effects of endoglin on the expression of H-Ras in transformed keratinocytes. We found that TGF-β1 increases the expression of H-Ras at both messenger RNA and protein levels. The TGF-β1-induced H-Ras promoter transactivation was Smad4 independent but mediated by the activation of the TGF-β type I receptor ALK5 and the Ras-mitogen-activated protein kinase (MAPK) pathway. Endoglin attenuated stimulation by TGF-β1 of both MAPK signaling activity and H-Ras gene expression. Moreover, endoglin inhibited the Ras/MAPK pathway in transformed epidermal cells containing an H-Ras oncogene, as evidenced by the levels of Ras-guanosine triphosphate, phospho-MAPK kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK) as well as the expression of c-fos, a MAPK downstream target gene. Interestingly, in spindle carcinoma cells, that have a hyperactivated Ras/MAPK pathway, endoglin inhibited ERK phosphorylation without affecting MEK or Ras activity. The mechanism for this effect is unknown but strongly depends on the endoglin extracellular domain. Because the MAPK pathway is a downstream mediator of the transforming potential of Ras, the effect of endoglin on the oncogenic function of H-Ras was assessed. Endoglin inhibited the transforming capacity of H-Ras(Q61K) and H-Ras(G12V) oncogenes in a NIH3T3 focus formation assay. The ability to interfere with the expression and oncogenic potential of H-Ras provides a new face of the suppressor role exhibited by endoglin in H-Ras-driven carcinogenesis.     

Ras/MAPK signaling from endomembranes

Signal transduction along the Ras/MAPK pathway has been generally thought to take place at the plasma membrane. It is now evident that the plasma membrane is not the only platform capable of Ras/MAPK signal induction. Fusion of Ras with green fluorescent protein and the development of genetically encoded fluorescent probes for Ras activation have revealed signaling events on a variety of intracellular membranes including endosomes, the Golgi apparatus and the endoplasmic reticulum. Thus, the Ras/MAPK pathway is spatially compartmentalized within cells and this may afford greater complexity of signal output.     

The Ras-Raf-MEK-ERK pathway in the treatment of cancer

The mitogen activated protein kinases (MAPKs) are conserved proteins that regulate cell growth, division and death. Although activated in the cytosol, the MAPKs translocate to the nucleus upon activation and phosphorylate a large number of nuclear proteins. Investigating how Ras transmits extracellular growth signals, the MAPK pathway has emerged as the crucial route between membrane-bound Ras and the nucleus. The MAPK pathway represents a cascade of phosphorylation events including three pivotal kinases, namely Raf, MEK (MAP kinase kinase), and ERK (MAP kinase). These kinases present new opportunities for the development of novel anti-cancer drugs designed to be target-specific and probably less toxic than conventional chemotherapeutic agents. A number of drugs inhibiting Ras, Raf or MEK are currently under clinical investigation. This review addresses the rationale for targeting the MAP kinase pathway and the current status of various pharmacological approaches.     

Development of anticancer drugs targeting the MAP kinase pathway.

Since the discovery of the role of ras oncogenes in tumorigenesis, we have witnessed an explosion of research in the signal transduction area. In the quest to understand how Ras transmits extracellular growth signals, the MAP kinase (MAPK) pathway has emerged as the crucial route between membrane-bound Ras and the nucleus. The MAPK pathway encompasses a cascade of phosphorylation events involving three key kinases, namely Raf, MEK (MAP kinase kinase) and ERK (MAP kinase). This kinase cascade presents novel opportunities for the development of new cancer therapies designed to be less toxic than conventional chemotherapeutic drugs. Furthermore, as a signal transduction-based approach to cancer treatment, inhibition of any one of these targets has the potential for translational pharmacodynamic evaluation of target suppression. The rationale for targeting the MAP kinase pathway will be reviewed here along with a discussion of various pharmacological approaches and the promise they hold for a new generation of anticancer drugs.     

Crosstalk between PI3K and Ras pathways via protein phosphatase 2A in human ovarian clear cell carcinoma

Hypoxia-inducible factor-1 (HIF-1) is one of the most promising pharmacological targets for all types of cancer, including ovarian cancer. Ovarian clear cell carcinoma (OCCC) has poor prognosis because of its insensitivity to chemotherapy. To elucidate the characteristics of this troublesome cancer, we examined HIF-1α expression under normoxia or hypoxia in various ovarian cancer cell lines. HIF-1α was highly expressed under normoxia only in RMG-1, an OCCC cell line. To examine whether HIF-1 is involved in the tumorigenesis of RMG-1 cells, we established HIF-1α-silenced cells, RMG-1HKD. The proliferation rate of RMG-1HKD cells was faster than that of RMG-1 cells. Furthermore, the activity of MEK/ERK in the Ras pathway increased in RMG-1HKD cells, whereas that of mTOR in the PI3K pathway did not change. Activation of the Ras pathway was attributable to the increase in phosphorylated MEK via PP2A inactivation. To confirm the crosstalk between the PI3K and Ras pathways in vivo, RMG-1 or RMG-1HKD cells were transplanted into the skin of nude mice with rapamycin (an inhibitor of mTOR), PD98059 (an inhibitor of MEK), or both. RMG-1HKD cells showed higher sensitivity to PD98059 than that observed in RMD-1 cells, whereas the combination therapy resulted in synergistic inhibition of both cells. These findings suggest that inhibition of HIF-1, a downstream target of mTOR in the PI3K pathway, activates the Ras pathway on account of the increase in MEK phosphorylation via PP2A inactivation, and the crosstalk between the 2 pathways could be applied in the combination therapy for HIF-1-overexpressing cancers such as OCCC.     

Differential regulation of MAP kinase cascade in human colorectal tumorigenesis

Hyper-activation of mitogen-activated protein kinase (MAPK) has recently been reported in several human cancers and activation of MAPK in those cancers may be associated with carcinogenesis through aberrant cell proliferation. To understand the roles of the MAPK pathway in colorectal tumorigenesis, we examined the status of extracellular signal-regulated protein kinases (ERK1/2) in 21 colorectal tumour specimens and compared it with that of paired normals. The specific MAPK activities were two- to tenfold lower in 71% (15 out of 21 cases) of colorectal tumours compared to those in paired normals. The individual MAPK kinase (MEK) correlated with MAPK activities (P = 0.006). Reduction of the MAPK and MEK activities in colorectal tumours was also observed in adenomas. These results suggested that down-regulation of the MAPK cascade may be caused by early genetic event(s) and that it may be related to the loss of normal growth control. Although MAPK activities were down-regulated both in adenomas and carcinomas, activities of the MAPKs in carcinomas were higher than those of paired adenomas. These results suggested that MAPK activities may be increased in the adenoma-to-carcinoma sequence and that it may play a role in the tumour progression. Observation of the differential regulation of MAPK activities in colorectal tumorigeneis suggested roles for the MAPK pathway in both positive and negative controls of cell growth.     

MEK inhibitor enhances the inhibitory effect of imatinib on pancreatic cancer cell growth

Imatinib mesylate (imatinib) inhibits the c-Kit-dependent tyrosine kinase activities and highly effective in the treatment of CML and GIST patients. Although pancreatic cancer is reported to express c-Kit, imatinib does not effectively inhibit pancreatic cancer cell growth at physiological concentrations. Therefore, we investigated the mechanism of resistance of pancreatic cancer to imatinib treatment. Imatinib inhibited growth of pancreatic cancer cell lines in concentration and time-dependent fashion regardless of c-Kit expression. However, 5 μM imatinib, which is almost a mean maximal plasma concentration in clinical setting, failed to suppress pancreatic cancer cell growth. Western blot analysis demonstrated that 5 μM imatinib treatment for 1 h activated the MEK–MAPK pathway and the activation was independent of Ras activation. Administration of 5 μM imatinib and 1 μM U0126 (MEK inhibitor) significantly suppressed pancreatic cell growth. Our results indicate that a combination therapy of imatinib and MEK inhibitor can be a new therapeutic strategy to suppress the progression of pancreatic cancer.     

Ras/MEK signaling suppresses Myc-dependent apoptosis in cells transformed by c-myc and activated ras

Cooperation of myc and activated ras has been suggested to cause malignant cell transformation but the mechanism is still unknown. Here we isolated a transformed cell line in which activation of c-Myc and Ras are independently controllable, and show that after establishment of the transformed state by c-myc and activated ras, removal of activated Ras initiates apoptosis that is dependent on c-Myc activity. Apoptosis is also initiated by an inhibitor of MEK (MAPK/ERK kinase), a kinase downstream of Ras, and apoptosis is blocked by activated Mek1. These results suggest that one of the conditions required for establishment of the transformed state is a block of apoptosis involving MEK activity. We tested the effect of MEK inhibition on cells transformed by various oncogenes. Suppression of apoptosis by MEK is not critical in general, but in cells transformed by c-myc plus a gene that activates the MAPK cascade it is necessary to avoid cell death. Activated Ras/MEK did not suppress c-myc-dependent apoptosis due to serum-limitation. Overexpression of chicken bcl-xL suppressed apoptosis under serum-limiting conditions, but not apoptosis initiated by Ras/MEK inhibition in cells transformed by myc and activated ras. Altogether, these results suggest the existence of a novel regulatory mechanism for myc-dependent apoptosis in certain transformed cells.     

Mitogen-activated protein kinase pathway-dependent tumor-specific survival signaling in melanoma cells through inactivation of the proapoptotic protein bad

Mitogen-activated protein kinase (MAPK) signaling regulates fundamental cellular functions including proliferation, differentiation, and survival. We have demonstrated previously that inhibiting MAPK signaling induces apoptosis in melanoma cells but not in normal melanocytes, suggesting that the MAPK pathway propagates essential survival signals in melanoma cells. Here, we report that the 90-kDa ribosomal S6 kinase (RSK), a downstream effector in the MAPK signaling cascade, phosphorylates and inactivates the Bcl-2 homology 3-only proapoptotic protein Bad, thereby mediating a MAPK-dependent tumor-specific survival signal in melanoma cells. The MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK)/RSK MAPK signaling module is constitutively hyperactivated, and Bad is maintained in its inactive state by phosphorylation at Ser(75) in a MEK/ERK/RSK-dependent manner in melanoma cells. In contrast, in normal melanocytes, Bad is highly phosphorylated at multiple residues (Ser(75), Ser(99), and Ser(118)) in a MAPK pathway-independent manner. Importantly, ectopic expression of a constitutively activated RSK mutant abrogates Bad activation and renders melanoma cells resistant to apoptosis induced by a MEK inhibitor. Furthermore, overexpressing alanine-substituted (S75A) Bad further sensitizes melanoma cells to MEK inhibitor-induced apoptosis. Our results suggest that the MAPK pathway mediates melanoma-specific survival signaling by differentially regulating RSK-mediated phosphorylation of the proapoptotic protein Bad and may present potentially selective therapeutic targets for the treatment of melanomas.     

B-Raf and C-Raf are required for Ras-stimulated p42 MAP kinase activation in Xenopus egg extracts

During mitosis, a select pool of MEK1 and p42/p44 MAPK becomes activated at the kinetochores and spindle poles, without substantial activation of the bulk of the cytoplasmic p42/p44 MAPK. Recently, we set out to identify the MAP kinase kinase kinase (MAPKKK) responsible for this mitotic activation, using cyclin-treated Xenopus egg extracts as a model system, and presented evidence that Mos was the relevant MAPKKK . However, a second MAPKKK distinct from Mos was readily detectable as well. Here, we partially purify this second MAPKKK and identify it as B-Raf. No changes in the activity of B-Raf were detectable during progesterone-induced oocyte maturation, after egg fertilization, or during the early embryonic cell cycle, arguing against a role for B-Raf in the mitotic activation of MEK1 and p42 MAPK. Ras proteins can bring about activation of MEK1 and p42 MAPK in extracts, and Ras may contribute to signaling from the classical progesterone receptor during oocyte maturation and from receptor tyrosine kinases during early embryogenesis. We found that both B-Raf and C-Raf, but not Mos, are required for Ras-induced MEK1 and p42 MAPK activation. These data indicate that two upstream stimuli, active Ras and active Cdc2, utilize different MAPKKKs to activate MEK1 and p42 MAPK.     

PAK1 primes MEK1 for phosphorylation by Raf-1 kinase during cross-cascade activation of the ERK pathway

The serine/threonine kinase Raf-1 acts downstream of Ras in the MAPK pathway leading to ERK activation in response to mitogens. Raf-1 has oncogenic potential, but is normally controlled by a complex interplay of inhibitory and activating mechanisms. Although Raf-1 is phosphorylated in unstimulated cells, mitogens cause its membrane recruitment by Ras and subsequent phosphorylation on additional sites. Some of these events modulate Raf-1 kinase activity while others determine interactions with other proteins. These changes regulate the ability of Raf-1 to phosphorylate its downstream targets MEK1 and MEK2. Rho family small G proteins act synergistically with Raf-1 to stimulate the ERK pathway by a cross-cascade mechanism that enhances MEK phosphorylation by Raf-1. Here we show that both Raf-1 and MEK1 are phosphorylated by PAK1 and that mutations at PAK1 phosphorylation sites in either protein prevent cross-cascade activation. In contrast, MEK1 activation by constitutively-active Raf-1 is refractory to mutations at PAK1 phosphorylation sites. Phosphorylation of MEK1 on serine 298 does not appear to regulate the interaction between Raf-1 and MEK1, but rather the ability of Raf-1 to phosphorylate MEK1 with which it is complexed in vivo. Our findings indicate that PAK1 primes MEK1 for activation by Raf-1 and imply another level of regulation in the ERK cascade.     

Targeting SHP2 sensitizes differentiated thyroid carcinoma to the MEK inhibitor

Pharmacologic targeting of components of the MAPK/ERK pathway in differentiated thyroid carcinoma (DTC) is often limited due to the development of adaptive resistance. However, the detailed mechanism of MEK inhibitor (MEKi) resistance is not fully understood. Here, MEKi-resistant models were constructed successfully, in which multiple receptor tyrosine kinases (RTKs) signaling pathways and Src-homology 2 domain-containing phosphatase 2 (SHP2) were activated in MEKi-resistant cells. Given the physiological role of SHP2 as the downstream target of many RTKs, we first found blockade of SHP2 enhanced the sensitivity to MEKi in constructed MEKi-resistant models. Interestingly, we also found that compared with MEKi treatment alone, MEKi in combination with an SHP2 inhibitor markedly suppressed the reactivation of the MEK/ERK pathway; thus, the addition of the SHP2 inhibitor significantly improved the antitumor effects of MEKi. The synergistic suppression of DTC upon treatment with both inhibitors was further confirmed in xenograft models and transgenic models. Thus, our data suggest that RTKs activation leads to reactivation of the MAPK pathway and resistance to MEKi in DTC, which is reversed by SHP2 blockade. As a novel active inhibitor of SHP2, SHP099 in combination with MEKi is a promising therapeutic approach for advanced DTC and MEKi-resistant one.