Raf Pathways Research Articles

Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond.

The identification of mutationally activated BRAF in many cancers altered our conception of the part played by the RAF family of protein kinases in oncogenesis. In this Review, we describe the development of BRAF inhibitors and the results that have emerged from their analysis in both the laboratory and the clinic. We discuss the spectrum of RAF mutations in human cancer and the complex interplay between the tissue of origin and the response to RAF inhibition. Finally, we enumerate mechanisms of resistance to BRAF inhibition that have been characterized and postulate how strategies of RAF pathway inhibition may be extended in scope to benefit not only the thousands of patients who are diagnosed annually with BRAF-mutated metastatic melanoma but also the larger patient population with malignancies harbouring mutationally activated RAF genes that are ineffectively treated with the current generation of BRAF kinase inhibitors.

AB0134 Affibody Molecules Inhibiting the Interaction between RAS and RAF Suppress the Proliferation and the Production of Inflammatory Mediators by Synovial Cells

BackgroundRas proteins are present in rheumatoid arthritis (RA) synovial tissues, and are preferentially expressed in the intimal lining layer. Activation of Ras effecter pathways in synovial tissues, including MAP kinases,...

Evidence for aldosterone-dependent growth of renal cell carcinoma.

The aim if this study was to investigate the hypothesis that K-RAS 4A is upregulated in a mineralocorticoid-dependent manner in renal cell carcinoma and that this supports the proliferation and survival of some renal cancers. Expression of the K-RAS in renal tumour tissues and cell lines was examined by real-time PCR and Western blot and mineralocorticoid receptor, and its gatekeeper enzyme 11β-hydroxysteroid dehydrogenase-2 was examined by immunocytochemistry on a tissue microarray of 27 cases of renal cell carcinoma. Renal cancer cells lines 04A018 (RCC4 plus VHL) and 04A019 (RCC4 plus vector alone) were examined for the expression of K-RAS4A and for the effect on K-RAS expression of spironolactone blockade of the mineralocorticoid receptor. K-RAS4A was suppressed by siRNA, and the effect on cell survival, proliferation and activation of the Akt and Raf signalling pathways was investigated in vitro. K-RAS4A was expressed in RCC tissue and in the renal cancer cell lines but K-RAS was downregulated by spironolactone and upregulated by aldosterone. Spironolactone treatment and K-RAS suppression both led to a reduction in cell number in vitro. Both Akt and Raf pathways showed activation which was dependent on K-RAS expression. K-RAS expression in renal cell carcinoma is at least partially induced by aldosterone. Aldosterone supports the survival and proliferation of RCC cells by upregulation of K-RAS acting through the Akt and Raf pathways.

Ras activation mediates WISP-1-induced increases in cell motility and matrix metalloproteinase expression in human osteosarcoma

WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matrix cellular proteins. Osteosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effect of WISP-1 on migration activity in human osteosarcoma cells is mostly unknown. In this study, we first found that the expression of WISP-1 in osteosarcoma patients was significantly higher than that in normal bone and corrected with tumor stage. Exogenous treatment of osteosarcoma cells with WISP-1 promoted cell motility and matrix metalloproteinase (MMP)-2 and MMP-9 expression. In addition, the Ras and Raf-1 inhibitor or siRNA abolished WISP-1-induced cell migration and MMP expression. On the other hand, activation of the Ras, Raf-1, MEK, ERK, and NF-κB signaling pathway after WISP-1 treatment was demonstrated, and WISP-1-induced expression of MMPs and migration activity were inhibited by the specific inhibitor, and mutant of MEK, ERK, and NF-κB cascades. Taken together, our results indicated that WISP-1 enhances the migration of osteosarcoma cells by increasing MMP-2 and MMP-9 expression through the integrin receptor, Ras, Raf-1, MEK, ERK, and NF-κB signal transduction pathway.

TNFα Modulates Fibroblast Growth Factor Receptor 2 Gene Expression through the pRB/E2F1 Pathway: Identification of a Non-Canonical E2F Binding Motif

Interactions between epithelium and mesenchyme during wound healing are not fully understood, but Fibroblast Growth Factors (FGFs) and their receptors FGFRs are recognized as key elements. FGFR2 gene encodes for two splicing transcript variants, FGFR2-IIIb or Keratinocyte Growth Factor Receptor (KGFR) and FGFR2-IIIc, which differ for tissue localization and ligand specificity. Proinflammatory cytokines play an essential role in the regulation of epithelial-mesenchymal interactions, and have been indicated to stimulate FGFs production. Here we demonstrated that upregulation of FGFR2 mRNA and protein expression is induced by the proinflammatory cytokines Tumor Necrosis Factor-α, Interleukin-1β and Interleukin 2. Furthermore, we found that TNFα determines FGFR2 transcriptional induction through activation of pRb, mediated by Raf and/or p38 pathways, and subsequent release of the transcription factor E2F1. Experiments based on FGFR2 promoter serial deletions and site-directed mutagenesis allowed us to identify a minimal responsive element that retains the capacity to be activated by E2F1. Computational analysis indicated that this element is a non-canonical E2F responsive motif. Thus far, the molecular mechanisms of FGFR2 upregulation during wound healing or in pathological events are not known. Our data suggest that FGFR2 expression can be modulated by local recruitment of inflammatory cytokines. Furthermore, since alterations in FGFR2 expression have been linked to the pathogenesis of certain human cancers, these findings could also provide elements for diagnosis and potential targets for novel therapeutic approaches.

Abstract 905: Design and synthesis of regorafenib analogues against signal transducers and activators of transcription 3 in hepatocellular carcinoma cells.

Abstract Background: Regorafenib has been approved for the treatment of metastatic colorectal cancer (m-RCC) by FDA. The major targets of regorafenib are membrane-bound and intracellular kinases including RET, VEGFR1, VEGFR2, VEGFR3, KIT, PDGFR-alpha, PDGFR-beta, FGFR1, FGFR2, TIE2, DDR2, Trk2A, Eph2A, RAF-1, BRAF, BRAFV600E, SAPK2, PTK5, and Abl pathways. In HCC, signal transducers and activators of transcription 3 (STAT3) is a key transcription factor involving in cell proliferation and survival in hepatocellular carcinoma cells, Targeting STAT3 by small molecule is a promising strategy in HCC therapy. Methods: We have designed and synthesized a series of regorafenib analogues without kinase inhibitory activity. These compounds are treated with and analyzed in terms of STAT3 activity in HCC cell lines. Also, Effects of regorafenib analogues induced cell death are evaluated. Results: We demonstrate that regorafenib and its analogues repress STAT3 phosphorylation, subsequently abrogate STAT3-regulated protein, Mcl-1 and cyclin D1 and further induce apoptosis in HCC cells. Overexpression of STAT3 in PLC-5 cells attenuates apoptotic effect with regorafenib and its analogues treatment, suggesting STAT3, at least, is a major target. Interestingly, the new analugues of regorafenib are devoid of kinase inhibitory activity which implies the involvement of other regulation factors. The activity of negative regulator of STAT3, such as protein tyrosine phosphatases, is under investigation. In summary, the new analogues provide a lead for the design of a new class of agents that inhibit STAT3 along and synergize with clinical drugs in HCC.(Supported by NSC 100-2325-B-010-007) Citation Format: Jung-Chen Su, Kuen-Feng Chen, Wen-Tien Tai, Jui-Wen Huang, Chung-Wai Shiau. Design and synthesis of regorafenib analogues against signal transducers and activators of transcription 3 in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 905. doi:10.1158/1538-7445.AM2013-905

Candida albicans primes TLR cytokine responses through a Dectin-1/Raf-1-mediated pathway.

The immune system is essential to maintain homeostasis with resident microbial populations, ensuring that the symbiotic host-microbial relationship is maintained. In parallel, commensal microbes significantly shape mammalian immunity at the host mucosal surface, as well as systemically. Candida albicans is an opportunistic pathogen that lives as a commensal on skin and mucosa of healthy individuals. Little is known about its capacity to modulate responses toward other microorganisms, such as colonizing bacteria (e.g., intestinal microorganisms). The aim of this study was to assess the cytokine production of PBMCs induced by commensal bacteria when these cells were primed by C. albicans. We show that C. albicans and β-1,3-glucan induce priming of human primary mononuclear cells and this leads to enhanced cytokine production upon in vitro stimulation with TLR ligands and bacterial commensals. This priming requires the β-1,3-glucan receptor dectin-1 and the noncanonical Raf-1 pathway. In addition, although purified mannans cannot solely mediate the priming, the presence of mannosyl residues in the cell wall of C. albicans is nevertheless required. In conclusion, C. albicans is able to modify cytokine responses to TLR ligands and colonizing bacteria, which is likely to impact the inflammatory reaction during mucosal diseases.

Melanoma adapts to RAF/MEK inhibitors through FOXD3-mediated upregulation of ERBB3

The mechanisms underlying adaptive resistance of melanoma to targeted therapies remain unclear. By combining ChIP sequencing with microarray-based gene profiling, we determined that ERBB3 is upregulated by FOXD3, a transcription factor that promotes resistance to RAF inhibitors in melanoma. Enhanced ERBB3 signaling promoted resistance to RAF pathway inhibitors in cultured melanoma cell lines and in mouse xenograft models. ERBB3 signaling was dependent on ERBB2; targeting ERBB2 with lapatinib in combination with the RAF inhibitor PLX4720 reduced tumor burden and extended latency of tumor regrowth in vivo versus PLX4720 alone. These results suggest that enhanced ERBB3 signaling may serve as a mechanism of adaptive resistance to RAF and MEK inhibitors in melanoma and that cotargeting this pathway may enhance the clinical efficacy and extend the therapeutic duration of RAF inhibitors.

Phase II trial of sorafenib in esophageal (E) and gastroesophageal junction (GEJ) cancer: Response and prolonged stable disease observed in adenocarcinoma.

91 Background: Sorafenib is a tyrosine kinase inhibitor targeting VEGFr, PDGFr, Raf and other pathways. Encouraging response and survival were observed in a phase II trial of sorafenib with chemotherapy in GE cancer (J Clin Oncol 27:2947;2010). We performed a phase II trial of single-agent sorafenib with the primary endpoint of progression-free survival (PFS). Secondary endpoints include response and toxicity. Methods: Patients (Pts) with measurable metastatic E and GEJ cancer with ≤3 prior chemotherapy regimens were treated with sorafenib 400 mg BID. CT scans were performed monthly for the first 2 months, then every 2 months. Results: Thirty-five patients have been accrued, with 34 evaluable; median age 62, 31 male, 4 female, median KPS 80%, E 25, GEJ 10, adenocarcinoma (AC) 30 squamous 5, median no. of prior therapies 2. Of 31 response-evaluable Pts, 1 (3%) ongoing complete response was noted (34+ months) in a Pt with E AC with biopsy-proven lymph node recurrence after chemoradiation and surgery; 23 Pts (74%) had stable disease. Median PFS is 3.7 months (95% CI 1.9 to 4 months), with median overall survival 8.9 months (95% CI 6.9 to 11.6 months). Four patients remain on treatment. Significant grade 3 toxicities included hand foot reaction (3 Pts), rash (1 Pt), dehydration (3 Pts) and fatigue (2 Pts). Twenty-seven of 33 tumors (82%) tested positive for phospho-ERK by immunohistochemistry. Conclusions: Encouraging activity in terms of PFS has been noted in this heavily pre-treated population. Updated data will be presented. Supported by a grant from Bayer. Clinical trial information: NCT00917462.

Thrombin-induced NF-κB activation and IL-8/CXCL8 release is mediated by c-Src-dependent Shc, Raf-1, and ERK pathways in lung epithelial cells

In addition to its functions in thrombosis and hemostasis, thrombin also plays an important role in lung inflammation. Our previous report showed that thrombin activates the protein kinase C (PKC)α/c-Src and Gβγ/Rac1/PI3K/Akt signaling pathways to induce IκB kinase α/β (IKKα/β) activation, NF-κB transactivation, and IL-8/CXCL8 expressions in human lung epithelial cells (ECs). In this study, we further investigated the mechanism of c-Src-dependent Shc, Raf-1, and extracellular signal-regulated kinase (ERK) signaling pathways involved in thrombin-induced NF-κB activation and IL-8/CXCL8 release. Thrombin-induced increases in IL-8/CXCL8 release and κB-luciferase activity were inhibited by the Shc small interfering RNA (siRNA), p66Shc siRNA, GW 5074 (a Raf-1 inhibitor), and PD98059 (a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor). Treatment of A549 cells with thrombin increased p66Shc and p46/p52Shc phosphorylation at Tyr239/240 and Tyr317, which was inhibited by cell transfection with the dominant negative mutant of c-Src (c-Src DN). Thrombin caused time-dependent phosphorylation of Raf-1 and ERK, which was attenuated by the c-Src DN. Thrombin-induced IKKα/β phosphorylation was inhibited by GW 5074 and PD98059. Treatment of cells with thrombin induced Gβγ, c-Src, and p66Shc complex formation in a time-dependent manner. Taken together, these results show for the first time that thrombin activates Shc, Raf-1, and ERK through Gβγ, c-Src, and Shc complex formation to induce IKKα/β phosphorylation, NF-κB activation, and IL-8/CXCL8 release in human lung ECs.

Transcriptional regulation of the interleukin-11 gene by oncogenic Ras

Interleukin-11 (IL-11), which belongs to a class of IL6-type cytokines, plays an important role in inflammation, motility and invasion in cancer. The ras mutation is frequently found in human cancer, but little is known regarding the transcriptional activation of the IL-11 gene by the Ras signal pathway in tumour cells. In this study, we investigated the role of Ras in the regulation of IL-11 using two different cell model systems: mouse NIH3T3 cells over-expressing oncogenic Ras with a tet-on system and Capan-1 human pancreatic carcinoma cells harbouring a K-ras mutation. We found that IL-11 expression was up-regulated at the transcriptional level by oncogenic Ras. Activation of the AP-1 response element, located between -153 and -30 in the 5'-regulatory region of the IL-11 gene, was necessary for oncogenic Ras-induced IL-11 promoter activation. AP-1 proteins, including Fra-1 and Fra-2, were up-regulated through the Raf/MEK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways by oncogenic Ras. Knockdown of Fra-1 by siRNA in NIH3T3 or Capan-1 cells strongly attenuated oncogenic Ras-induced IL-11 expression. Additionally, inhibition of JNK, p38 and Stat3 abrogated oncogenic Ras-induced IL-11 expression. These results suggest that both the PI3K and Raf pathways are necessary for the expression of IL-11 in oncogenic Ras-mutated cells, and that JNK, p38 and Stat3 also contribute to oncogenic Ras-induced IL-11 expression.

Prognostic Value of CD133 Caused by Mutant K-Ras and B-Raf—Letter

Kemper and colleagues (1) provided evidence that the expression of the cancer stem cell (CSC) marker CD133 is upregulated in colorectal tumors that have a hyperactivated Ras–Raf–MEK–ERK pathway and is, therefore, related to mutations in K-Ras or B-Raf. Thus, CD133 expression is not indicative for CSC numbers but rather for the mutation or activity status of the Ras–Raf pathway.In our opinion, some key points should be discussed in more detail. First of all, the choice to focus on stage II colon cancer is valuable, as adjuvant treatment is a matter of debate in this subset. However, Kemper and colleagues do not include all the clinical variables with already known prognostic value, particularly lymphovascular invasion and the number of examined nodes. Therefore, it is difficult to argue for the additive value of CD133 evaluation in the prognostic stratification of stage II patients beyond conventional clinicopathologic features.More importantly, CD133 levels seem to retain more prognostic information than that enclosed in the K-Ras/B-Raf mutational status. In fact, neither K-Ras nor B-Raf mutations [as well as microsatellite instability (MSI) status] are associated with relapse-free survival (RFS) at univariate analysis, whereas an analysis comprising all the examined variables is not provided. As reviewed in the work of Sinicrope and Sargent (2), K-Ras and B-Raf mutations do not predict RFS, whereas MSI status is convincingly associated with tumor recurrence in stage II colon cancer. The prognostic value of B-Raf mutation in colorectal cancer seems confined to the metastatic setting, as shown by the association with overall survival rather than RFS in larger series of stage II–III cases (2). Unfortunately, Kemper and colleagues do not evaluate the role of MSI in B-Raf mutant patients, as the literature suggests that prognosis may be particularly poor in MSI-low or stable B-Raf mutant tumors (2).From a biologic perspective, presented data indicate that K-Ras and B-Raf mutations contribute to the regulation of CD133 expression, but they are probably not the only actors. Additional investigation, therefore, is needed to reach the conclusion that the link between CD133 levels and prognosis is only dependent on the Ras–Raf axis. Moreover, factors other than somatic mutations contribute to the regulation of the Ras–Raf pathway, as proven by the recent identification of a subpopulation of B-Raf wild-type tumors with poor prognosis and gene expression patterns similar to those of B-Raf mutant tumors (independently of the K-Ras status; ref. 3). Furthermore, it is unlikely that a single marker (such as CD133) covers all the complexity and dynamics of CSCs (4). For example, Huang and colleagues showed that only the CD133+/CD44+ fraction predicts metastatic risk in colorectal cancer (5). We suggest that CSC-specific signaling pathways, rather than putative CSC surface markers, may be more useful prognostic and predictive biomarkers.In conclusion, factors other than K-Ras/B-Raf mutational status may be involved in CD133 regulation and, more intriguingly, additional CSC parameters may strengthen the value of CD133 in clinics beyond already available prognostic factors. We are therefore indebted to the authors for their report, but this is just a glimpse into the dark mysteries of CSCs in colorectal cancer.See the Response, p. 4474No potential conflicts of interest were disclosed.

Candida albicans Infection Affords Protection against Reinfection via Functional Reprogramming of Monocytes

Immunological memory in vertebrates is often exclusively attributed to T and B cell function. Recently it was proposed that the enhanced and sustained innate immune responses following initial infectious exposure may also afford protection against reinfection. Testing this concept of "trained immunity," we show that mice lacking functional T and B lymphocytes are protected against reinfection with Candida albicans in a monocyte-dependent manner. C. albicans and fungal cell wall β-glucans induced functional reprogramming of monocytes, leading to enhanced cytokine production in vivo and in vitro. The training required the β-glucan receptor dectin-1 and the noncanonical Raf-1 pathway. Monocyte training by β-glucans was associated with stable changes in histone trimethylation at H3K4, which suggests the involvement of epigenetic mechanisms in this phenomenon. The functional reprogramming of monocytes, reminiscent of similar NK cell properties, supports the concept of "trained immunity" and may be employed for the design of improved vaccination strategies.

The PP242 Mammalian Target of Rapamycin (mTOR) Inhibitor Activates Extracellular Signal-regulated Kinase (ERK) in Multiple Myeloma Cells via a Target of Rapamycin Complex 1 (TORC1)/ Eukaryotic Translation Initiation Factor 4E (eIF-4E)/RAF Pathway and Activation Is a Mechanism of Resistance

Activation of PI3-K-AKT and ERK pathways is a complication of mTOR inhibitor therapy. Newer mTOR inhibitors (like pp242) can overcome feedback activation of AKT in multiple myeloma (MM) cells. We, thus, studied if feedback activation of ERK is still a complication of therapy with such drugs in this tumor model. PP242 induced ERK activation in MM cell lines as well as primary cells. Surprisingly, equimolar concentrations of rapamycin were relatively ineffective at ERK activation. Activation was not correlated with P70S6kinase inhibition nor was it prevented by PI3-kinase inhibition. ERK activation was prevented by MEK inhibitors and was associated with concurrent stimulation of RAF kinase activity but not RAS activation. RAF activation correlated with decreased phosphorylation of RAF at Ser-289, Ser-296, and Ser-301 inhibitory residues. Knockdown studies confirmed TORC1 inhibition was the key proximal event that resulted in ERK activation. Furthermore, ectopic expression of eIF-4E blunted pp242-induced ERK phosphorylation. Since pp242 was more potent than rapamycin in causing sequestering of eIF-4E, a TORC1/4E-BP1/eIF-4E-mediated mechanism of ERK activation could explain the greater effectiveness of pp242. Use of MEK inhibitors confirmed ERK activation served as a mechanism of resistance to the lethal effects of pp242. Thus, although active site mTOR inhibitors overcome AKT activation often seen with rapalog therapy, feedback ERK activation is still a problem of resistance, is more severe than that seen with use of first generation rapalogs and is mediated by a TORC1- and eIF-4E-dependent mechanism ultimately signaling to RAF.

Mutations in the Ras–Raf Axis Underlie the Prognostic Value of CD133 in Colorectal Cancer

High expression of cancer stem cell (CSC) marker CD133 has been used as a predictor for prognosis in colorectal cancer (CRC), suggesting that enumeration of CSCs, using CD133, is predictive for disease progression. However, we showed recently that both CD133 mRNA and protein are not downregulated during differentiation of colon CSCs, pointing to an alternative reason for the prognostic value of CD133. We therefore set out to delineate the relation between CD133 expression and prognosis. A CRC patient series was studied for expression of CD133 and other CSC markers by microarray and quantitative PCR analysis. In addition, several common mutations were analyzed to determine the relation with CD133 expression. CD133 mRNA expression predicted relapse-free survival in our patient series, whereas several other CSC markers could not. Moreover, no correlation was found between expression of other CSC markers and CD133. Interestingly, high CD133 expression was related to mutations in K-Ras and B-Raf, and inhibition of mutant K-Ras or downstream mitogen-activated protein kinase kinase (MEK) signaling decreases CD133 expression. In addition, an activated K-Ras gene expression signature could predict CD133 expression in our patient set as well as data sets of other tumor types. CD133 expression is upregulated in CRC tumors that have a hyperactivated Ras-Raf-MEK-ERK pathway and is therefore related to mutations in K-Ras or B-Raf. As mutations in either gene have been related to poor prognosis, we conclude that CD133 expression is not indicative for CSC numbers but rather related to the mutation or activity status of the Ras-Raf pathway.

Abstract 2706: Phase 2 study with pharmacokinetics (PK) analysis of sorafenib and vinorelbine in metastatic breast cancer

Abstract Background Sorafenib (S) is an oral inhibitor of the Raf, VEGFR and PDGFR pathways that contribute to chemoresistance in cancer cells. The combination of S with chemotherapy agents (capecitabine, paclitaxel) in metastatic breast cancer (BC) resulted in improved progression-free survival. In preclinical models of BC, favorable effects were noted combining S and vinorelbine (V). S and V are both metabolized through hepatic isoenzymes CYP3A, with the potential of PK interactions. A previous phase 1b study documented the feasibility of this combination. We aimed to define the activity and safety of this regimen in advanced BC and to investigate PK interactions of the 2 drugs at full doses. Methods Eligible patients were affected by Her2 negative, chemotherapy-naive metastatic BC, with measurable (RECIST) disease, adequate renal, liver and bone marrow functions and LVEF Δ50%. Treatment included V (30 mg/m2 days 1, 8 every 21) + S (400 mg bid continuously), with dose reductions for grade 2-4 toxicities. Patients still benefiting after 8 cycles of treatment could continue on single-agent S. Six patients underwent PK analysis, starting treatment with S on day 4 of the first cycle, allowing for comparison of PK of each agent (V and S) administered alone versus during concomitant treatment with the other agent. Results 27 evaluable patients started on treatment, median age 57 (35-78); they received a median of 8 cycles to date (1-28), with 4 patients still on treatment. 44% of patients required at least one dose reduction for toxicity. One patient died of complicated sepsis after febrile neutropenia. Other significant toxicities included: febrile neutropenia (8%), uncomplicated Grade 3 (G3)-G4 neutropenia (60%); G3 fatigue (20%), hand-foot syndrome (12%), diarrhea (4%), enteritis (4%), hypertension (4%), asymptomatic increase of AST/ALT (16%), amylase (12%), lipase (8%), GGT (4%); G2 decrease in LVEF (4%), rash (16%), alopecia (20%). 33% of patients had a partial response, 85% experienced a clinical benefit (including stable disease after at least 4 cycles of treatment). Median progression-free survival was 5.5 months (95% CI 4.6-7.5). PK analysis showed no statistically significant interaction between S and N. Also the conversion of S to its metabolite S-N-oxide was not affected by the concomitant treatment with V. A non-significant trend was noticed for higher Cmax/AUC of V in the first 30 minutes after infusion when administered with ongoing treatment with S. Conclusions Combining S with V is feasible, but not devoid of toxicity, for which frequent dose reductions are necessary. A PK interaction could not be ruled out with the small sample size analyzed. More frail patients should start at a lower dose of both agents and we recommend close monitoring for the first 2 cycles, including amylase and lipase. Promising efficacy of this combination was observed, with a very high rate of disease control. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2706. doi:1538-7445.AM2012-2706

Abstract PL04-02: Ras and Raf signaling: From biology to therapeutics

Abstract The protein kinase BRAF is mutated in about 50% of human melanomas and the small G-protein NRAS is mutated in another 20% of cases. We have developed mouse models of melanoma driven by oncogenic BRAF, NRAS and KRAS. These studies have established that acquisition of BRAF, NRAS or KRAS mutations can be founder events in melanomagenesis and we are currently using these models to investigate gene-gene and gene-environment interactions in cutaneous melanoma. We are also investigating other targets of BRAF in melanoma cells and have shown that BRAF drives melanoma cell migration and invasion by down-regulating expression of the cyclic GMP phosphodiesterase PDE5A. 2011 was a landmark year in melanoma research with the registration of vemurafenib and ipilimumab, drugs that show clinical benefit in patients with advanced disease. Critically, BRAF inhibitory drugs achieve objective clinical responses in about 80% of melanomas that harbour mutations in BRAF. Surprisingly however, these drugs also drive paradoxical activation of CRAF in melanoma cells that harbour mutations in NRAS, or in cells in which RAS is activated due to increased upstream signalling. This is due to the induction of BRAF/CRAF heterodimers and CRAF homodimers and importantly, activation of the pathway by this paradox can drive tumourigenesis in some circumstances. We have used a two-stage skin carcinogenesis model to show that BRAF inhibitors are not tumour promoters pre se, but rather that they work by accelerating the growth of tumours carrying pre-existing mutations in HRAS. Thus, we conclude that BRAF inhibitors appear to accelerate the inevitable in susceptible patients, but do not induce these lesions de novo. Notably, concurrent administration of a MEK inhibitor with a BRAF drug blocks the development of these skin lesions, providing a rationale for combining BRAF and MEK inhibitors in melanoma patients. Unexpectedly, we have also found that the ABL/CKIT inhibitor nilotinib also drives paradoxical activation of the RAF pathway because it possesses weak off-target activity against BRAF and CRAF. The consequence of this paradoxical activation is that nilotinib provides a survival advantage to chronic myeloid leukaemia (CML) cells that are resistant to nilotinib, but also provides an Achilles heel to these cells because they become dependent on MEK signalling in the presence of nilotinib. Thus, MEK inhibitors cooperate with nilotinib to drive synthetic lethality in nilotinib-resistant CML cells. Thus, paradoxical activation of the RAF proteins by small molecular weight drugs can have important consequences to patient responses, creating specific clinical problems in some circumstances, but therapeutic opportunities in others. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr PL04-02. doi:1538-7445.AM2012-PL04-02

Active surrender

Active surrender

Cell Development Pathways Follow from a Principle of Extreme Fisher Information

Background: In a normally developing eukaryote, information arrives at the cell membrane in the form of a ligand that binds to a protein receptor. This initiates a cascade of biochemical events causing one or more proteins to subsequently traverse the cell cytoplasm to the nucleus. This defines a communication channel. What does it accomplish? Method: The protein traversals transfer to the nucleus maximum Fisher information about the spatial and temporal coordinates of the ligand binding sites. This hypothesis implies a cell model of fast, largely-directed, protein movement dominated by Coulomb interaction with intracellular electric fields. It makes the following predictions: (1) Very high intracellular electric field strengths, typically tens of millions of volts/meter (2) A central role for negative charges added to proteins by phosphorylation, in promoting their Coulomb force-dominated motion toward the nucleus; (3) The dominance of protein pathways consisting of from 1-4 proteins, e.g. the RAF, RAS and MEK pathways; (4) A predicted fast response (2,800 proteins/ ms ) of cells to sudden trauma such as wounds; (5) A predicted 4nm size (9) for the EGFR protein. (6) Logic mechanisms in the nucleus for optimally deconvolving spatial and temporal binding site values from the inflowing messenger proteins. Results: Predictions (1-5) are supported by laboratory observations. Conclusions: Living systems achieve stably ordered and complex states by maintaining extreme levels of Fisher information. The attained order values increase from cancers to prokaryotes to eukaryotes to multicellular organisms. In eukaryotes this fosters maximally high protein flux rates at the nucleus which, in turn, optimize wired-in intranuclear logic mechanisms for processing this, and other, temporal and spatial information.

CCL2 increases MMP-9 expression and cell motility in human chondrosarcoma cells via the Ras/Raf/MEK/ERK/NF-κB signaling pathway

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family that is associated with the disease status and outcomes of cancers. However, the effect of CCL2 on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that CCL2 increased the migration and expression of matrix metalloproteinase (MMP)-9 in human chondrosarcoma cells. CCL2-mediated migration and MMP-9 up-regulation were attenuated by CCR2, Ras, Raf-1, and MEK inhibitor. Activation of the Ras, Raf-1, MEK, ERK, and NF-κB signaling pathway after CCL2 treatment was demonstrated, and CCL2-induced expression of MMP-9 and migration activity were inhibited by the specific inhibitor, and mutant of Ras, Raf-1, MEK, ERK, and NF-κB cascades. Taken together, our results indicated that CCL2 enhances the migration of chondrosarcoma cells by increasing MMP-9 expression through the CCR2 receptor, Ras, Raf-1, MEK, ERK, and NF-κB signal transduction pathway.