RAF protein-serine/threonine kinases: Structure and regulation

A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.     

Characterization of a maize ERF gene, <Emphasis Type="Italic">ZmERF1,</Emphasis> in hormone and stress responses

ERFs are downstream component in ethylene signaling pathway and involved in plant’s abiotic stress response. The specific role of ERFs under stress and the molecular mechanism underlying the signaling cross talk still need to be elucidated. This study describes the isolation and characterization of ZmERF1 promoter. There were many cis-regulatory elements related to stress responses in the ZmERF1 promoter sequence. ZmERF1 could be highly induced by ABA and ethylene treatment in maize, suggesting that it might be at the crossroads of multiple hormone signaling pathways. Furthermore, ZmERF1 transgenic Arabidopsis lines (35S::ZmERF1) showed higher salt-tolerant, drought- and heat resistance. Consistently, tolerance-related genes were up-regulated in 35S::ZmERF1 lines compared with the WT plants in Arabidopsis. Overall, ZmERF1 might play an important role in plant resistance to a coercive environment by mediating various physiological processes via ethylene and ABA signaling pathways.     

Induction of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Resistance to Pathogenic Bacteria by Lipopolysaccharide and Salicylic Acid

The effects of combined treatment with an elicitor (lipopolysaccharide) and a signaling molecule (salicylic acid) on the disease resistance of wild-type (Col-0) and mutant Arabidopsis thaliana L. plants have been compared. The mutant lines used were jin1 (with impaired jasmonate signaling), npr1 (lacking expression of pathogen-dependent PR genes), and NahG (expressing an active bacterial salicylate hydroxylase transgene). The lipopolysaccharide was isolated from a saprophytic strain (8614) of Pseudomonas aeruginosa bacteria. Treatment of A. thaliana seeds with a composite preparation (lipopolysaccharide and salicylic acid–SA) increased the resistance of seedlings to a subsequent infection by the pathogenic 9096 strain of P. aeruginosa bacteria. The protective effect was more pronounced in jin1 mutant seedlings, which was indicative of the possible compensation of jasmonate signaling impairment due to activation of the SA-dependent signaling pathway. We concluded that a preparation composed of an elicitor and a signaling molecule could affect regulatory mechanism functioning in a plant cell and, in particular, compensate for the absence of a certain signaling pathway by activating another.     

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.     

Determination of <Emphasis Type="Italic">EGFR</Emphasis> gene somatic mutations in tissues and plasma of patients with non-small cell lung cancer

Activating mutations in the EGFR gene influence cell proliferation, angiogenesis, and increases metastatic ability of non-small cell lung cancer (NSCLC) cells; they have a significant impact on the choice of medical therapy of NSCLC. The use of targeted therapy with tyrosine kinase inhibitors requires performance of appropriate genetic tests in NSCLC patients. The aim of this study was to develop a real-time PCRbased diagnostic test-system for rapid and cost-effective analysis of EGFR mutations in paraffin blocks and plasma and to perform comparative estimation of diagnostic characteristics features of real-time wild type blocking PCR and digital PCR. The study included 156 patients with different degrees of lung adenocarcinoma differentiation. A simple and efficient real-time PCR-based method for detection of L858R activating mutation and del19 deletion in the EGFR gene in DNA isolated from paraffin blocks or blood has been developed. The test system for EGFR mutations has been validated using 411 samples of paraffin blocks. The proposed system demonstrated high efficiency for DNA testing from paraffin blocks: a concordance with results of testing by means a Therascreen® EGFR RGQ PCR Kit (Qiagen, Germany) was 100%. Applicability of this test system has been also demonstrated for detection of mutations in plasma.     

Activity of the Heat Shock Protein 90 Inhibitor Ganetespib in Melanoma

Heat shock protein 90 (HSP90) is involved in the regulation of diverse biological processes such as cell signaling, proliferation and survival, and has been recently recognized as a potential target for cancer therapy. Ganetespib is a potent ATP competitive inhibitor of HSP90. Ganetespib downregulated the expression of multiple signal transducing molecules including EGFR, IGF-1R, c-Met, Akt, B-RAF and C-RAF, resulting in pronounced decrease in phosphorylation of Akt and Erk1/2 in a panel of five cutaneous melanoma cell lines including those harboring B-RAF and N-RAS mutations. Ganetespib exhibited potent antiproliferative activity on all five of these cell lines, with IC50 values between 37.5 and 84 nM. Importantly, Ganetespib is active on B-RAF mutated melanoma cells that have acquired resistance to B-RAF inhibition. Ganetespib induced apoptosis and cell cycle arrest at G1 and/or G2/M phase. Ganetespib induced cell cycle arrest was accompanied by altered expression of cyclin-dependent kinase inhibitor (CDKI) p21^Cip1 and p27^Kip1, cyclins B1, D1 and E, and/or cyclin-dependent kinases 1, 2 and 4. HSP90 is functionally important for melanoma cells and HSP90 inhibitors such as ganetespib could potentially be effective therapeutics for melanoma with various genetic mutations and acquired resistance to B-RAF inhibition.     

<Emphasis Type="Italic">In vivo</Emphasis> RNAi screen identifies candidate signaling genes required for collective cell migration in <Emphasis Type="Italic">Drosophila</Emphasis> ovary

Collective migration of loosely or closely associated cell groups is prevalent in animal development, physiological events, and cancer metastasis. However, our understanding of the mechanisms of collective cell migration is incomplete. Drosophila border cells provide a powerful in vivo genetic model to study collective migration and identify essential genes for this process. Using border cell-specific RNAi-silencing in Drosophila, we knocked down 360 conserved signaling transduction genes in adult flies to identify essential pathways and genes for border cell migration. We uncovered a plethora of signaling genes, a large proportion of which had not been reported for border cells, including Rack1 (Receptor of activated C kinase) and brk (brinker), mad (mother against dpp), and sax (saxophone), which encode three components of TGF-β signaling. The RNAi knock down phenotype was validated by clonal analysis of Rack1 mutants. Our data suggest that inhibition of Src activity by Rack1 may be important for border cell migration and cluster cohesion maintenance. Lastly, results from our screen not only would shed light on signaling pathways involved in collective migration during embryogenesis and organogenesis in general, but also could help our understanding for the functions of conserved human genes involved in cancer metastasis.     

<Emphasis Type="Italic">OXI1</Emphasis> kinase plays a key role in resistance of Arabidopsis towards aphids (<Emphasis Type="Italic">Myzus persicae</Emphasis>)

Plants have co-evolved with a diverse array of pathogens and insect herbivores and so have evolved an extensive repertoire of constitutive and induced defence mechanisms activated through complex signalling pathways. OXI1 kinase is required for activation of mitogen-activated protein kinases (MAPKs) and is an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses. Furthermore, changes in the levels of OXI1 appear to be crucial for appropriate signalling. Callose deposition also plays a key role in defence. Here we demonstrate, for the first time, that OXI1 plays an important role in defence against aphids. The Arabidopsis mutant, oxi1-2, showed significant resistance both in terms of population build-up (p?<?0.001) and the rate of build-up (p?<?0.001). Arabidopsis mutants for β-1,3-glucanase, gns2 and gns3, showed partial aphid resistance, significantly delaying developmental rate, taking two-fold longer to reach adulthood. Whilst β-1,3-glucanase genes GNS1, GNS2, GNS3 and GNS5 were not induced in oxi1-2 in response to aphid feeding, GNS2 was expressed to high levels in the corresponding WT (Col-0) in response to aphid feeding. Callose synthase GSL5 was up-regulated in oxi1-2 in response to aphids. The results suggest that resistance in oxi1-2 mutants is through induction of callose deposition via MAPKs resulting in ROS induction as an early response. Furthermore, the results suggest that the β-1,3-glucanase genes, especially GNS2, play an important role in host plant susceptibility to aphids. Better understanding of signalling cascades underpinning tolerance to biotic stress will help inform future breeding programmes for enhancing crop resilience.     

Hormone resistance and neuroendocrine differentiation due to accumulation of genetic lesions during clonal evolution of prostate cancer

Progression of malignant tumors is largely due to clonal evolution of the primary tumor, clones acquiring different sets of molecular genetic lesions. Lesions can confer a selective advantage in proliferation rate or metastasis on the tumor cell population, especially if developing resistance to anticancer therapy. Prostate cancer (PCa) provides an illustrative example of clinically significant clonal evolution. The review considers the genetic alterations that occur in primary PCa and the mechanism whereby hormone-refractory PCa develops on hormone therapy, including mutations and alternative splicing of the androgen receptor gene (AR) and intratumoral androgen synthesis. Certain molecular genetic lesions determine resistance to new generation inhibitors (AR mutations that block the antagonist effect or allow other hormones to activate the receptor) or lead to neuroendocrine differentiation (repression of the AR signaling pathway, TP53 mutations, and amplification of the AURKA or MYCN oncogene). Multistep therapy based on the data about somatic mutations associated with progression and metastasis of the primary tumor can be expected to significantly improve the survival of patients with advanced PCa in the nearest future.     

Cloning of the doding cDNA sequence of the gene for Csk tyrosine kinase from human lymphocytes

Tyrosine kinases of the Csk family play an important role in cell growth regulation and normal cell differentiation. They are also involved in carcinogenesis as oncoproteins. The main function of these tyrosine kinases is phosphorylation of tyrosine kinases of the Src family at their C-terminal regions to negatively regulate their activity. Disturbance of csk expression increases the Src tyrosine kinase activity. The full-length coding sequence of the csk cDNA was cloned from human lymphocytes. The 1624-bp cDNA consists of 12 exons and encodes a protein that has conserved SH2 and SH3 domains and is similar to human Csk tyrosine kinase by 99%. The full-length cDNA can be used to analyze the csk structure in normal or illdefined human cells.