Egfr Activation Protocol

ADP-Glo Kinase Assay Application Notes TYROSINE KINASE SERIES: EGFR EGFR Kinase Assay. By Hicham Zegzouti, Ph.D., Jolanta Vidugiriene, Ph.D., and Said A.

The structure and function of the epidermal growth factor receptor EGFR is evolutionarily conserved from Caenorhabditis elegans to Homo sapiens Aroian et al., 1990 and its activity regulates the proliferation, motility, and differentiation of many different cell types Sibilia and Wagner, 1995 ; Threadgill et al., 1995 . Binding of any one of at least five ligands activates the intrinsic tyrosine kinase domain of the EGFR van der Geer et al., 1994 , which phosphorylates itself and activates other members of the EGFR family, such as HER2 Stern and Kamps, 1988 ; van der Geer et al., 1994 . Receptor phosphotyrosine residues act as nucleation sites for additional proteins such as Shc, Grb2, mSOS, ras-GAP, phospholipase C-γ, Eps8, and c-Cbl Rozakis-Adcock et al., 1992 ; Fazioli et al., 1993 ; van der Geer et al., 1994 ; Levkowitz et al., 1998 . These receptor signaling partners are activated by allosteric effects or by tyrosine phosphorylation, leading to recruitment of additional signaling molecules van der Geer et al., 1994 . Downstream kinase cascades and specific protein-protein assemblages can, in turn, determine cell type-specific responses Tan and Kim, 1999 .

Activated EGFR are rapidly internalized by coated pits, sorted through early endosomes, and ultimately degraded in lysosomes by a process generally known as receptor down-regulation Wiley et al., 1991 ; Sorkin and Waters, 1993 . G-protein coupled receptors, as well as other receptor tyrosine kinases, are also down-regulated after ligand activation Sorkin and Waters, 1993 ; Kallal et al., 1998 . Although degradation is the ultimate fate of internalized receptors, the rate of receptor degradation is much slower than their rate of internalization. Thus, substantial intracellular pools of receptors and ligands can accumulate Wiley et al., 1985 . It is clear that receptors are initially activated at the plasma membrane, but it is much less certain whether internalized receptors remain active until they are degraded. It is also unknown whether signals from internalized receptors are qualitatively different from those generated at the cell surface.

For more than a decade, investigators have debated the existence of signaling endosomes. Experiments with rat liver have demonstrated that, after the administration of a bolus of EGF, intracellular EGFR are associated with Shc, Grb2, and mSOS Di Guglielmo et al., 1994 . These signaling cofactors are thought to be responsible for initiating signals at the cell surface van der Geer et al., 1994 . Additionally, other receptor substrates, such as c-src and rho-B, are enriched in endosomes Adamson et al., 1992 ; Kaplan et al., 1992 . The strongest evidence supporting the signaling endosome hypothesis comes from recent genetic and biochemical experiments with the EGFR and the β-adrenergic receptor. Schmid and colleagues used a conditional dynamin mutant to block EGFR endocytosis, resulting in specific signal transduction pathways being up-regulated and others being attenuated Vieira et al., 1996 . In similar experiments with the β-adrenergic receptor, endocytosis was inhibited with the use of both the nonspecific conditional dynamin mutation and a specific mutation in β-arrestin. This resulted in inhibition of mitogen-activated protein kinase activation Daaka et al., 1998 ; Ahn et al., 1999 . Together, these data suggest that specific signals can arise from the endosomal compartment.

Despite the positive evidence, it has been argued that EGFR signal transduction is primarily restricted to the cell surface Fiore and Gill, 1999 . To a large extent, this idea is based on the correlation between low rates of EGFR internalization and cell transformation Wells et al., 1990 ; Huang et al., 1997 . Supporting this argument is the observation that v-Cbl transforms cells at least in part by shunting EGFR back to the cell surface Levkowitz et al., 1998 . These data, however, do not directly rule out the possibility that signal transduction can arise from endosomes; nor do they separate the effects of inhibiting receptor endocytosis from the effects of inhibiting ligand or receptor degradation. Endosomes could still make up an important signaling compartment.

A major difficulty in evaluating the role of endosomal signaling is the low sensitivity of current techniques. In general, one must isolate endosomal compartments at different times after ligand stimulation and evaluate their composition Wada et al., 1992 . Because of the low yield and time-consuming nature of this approach, previous studies have been restricted to abundant tissues, such as rat liver, or transformed cells that overexpress receptors or specific signaling components Levkowitz et al., 1998 ; Xue and Lucocq, 1998 . Although these studies have been informative, they have necessary limitations. Rat liver is not a physiologically important target of EGFR action and overexpression of receptors or signaling molecules can lead to altered trafficking or function. These technical issues have made it difficult to determine whether endosomal signaling is a normal consequence of EGFR activation or is restricted to specific experimental systems.

To investigate the role of EGFR trafficking in its biological actions, we have used responsive human mammary epithelial cells HMEC. Genetic and biochemical studies in mice have shown that normal EGFR function is critical for the development of the mammary epithelium Fowler et al., 1995 ; Xie et al., 1997 . In vitro, blocking the EGFR in HMEC leads to cell cycle arrest as well as inhibition of cell migration and organization Stampfer et al., 1993 ; Wiley et al., 1998 ; Dong et al., 1999 . Importantly, HMEC normally express high levels of EGFR, facilitating biochemical studies Bates et al., 1990 ; Burke and Wiley, 1999 . To investigate EGFR trafficking, we developed a new biochemical technique to isolate activated EGFR within endosomes with the use of a reversibly biotinylated nonantagonistic anti-EGFR antibody. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR to follow activated EGFR by immunofluorescence techniques. With the use of these approaches, we observed that the pattern of EGFR association with substrates and adaptor proteins changed as the EGFR moved from the cell surface through the endosomal compartment. In addition, we found that internalized EGFR lost both phosphotyrosine and associated ligand before degradation. Our results suggest that endosomes make up a major site of regulated EGFR signaling in responsive cells and that ligand loss is the proximal cause of attenuated receptor signaling.

Pancreatic ductal adenocarcinoma PDA has a 5-year survival rate of less than 5 Jemal et al., 2008. To date, chemo- and radiotherapy regimens have failed to increase patient survival times significantly. Resistance to chemotherapy and molecularly targeted therapies is thought to underlie these treatment failures Giovannetti et al., 2006. Recent work from our group has identified HB-EGF as having a key role in PDA chemoresistance Wang et al., 2007a, 2007b. HB-EGF binds to EGFR and HER4 leading to activation of several signal transduction cascades that have critical function in diverse cell fates including development, proliferation, differentiation and migration Nishi and Klagsbrun, 2004. HB-EGF expression has been implicated in tumor initiation and progression and it is overexpressed in many tumors including pancreatic, hepatocellular, colonic and bladder cancers Raab and Klagsbrun, 1997. Studies both in vitro and in human tumor specimens have additionally identified involvement of HB-EGF in resistance to a wide variety of clinically used molecular and genotoxic agents Suganuma et al., 2003; Wang et al., 2007a. These findings show that HB-EGF is not only a potent inducer of tumor growth but is also a survival factor involved in response to cellular stress.

Along with others, we have shown that proteasome inhibitors have broad in vitro and in vivo antitumor activity and can overcome chemoresistance in a number of cancer models including PDA Milano et al., 2007; McConkey and Zhu, 2008. This activity has been predominantly attributed to their ability to inhibit NF-κB, dysregulate the cell cycle, induce ER stress and stabilize proapoptotic proteins Milano et al., 2007; McConkey and Zhu, 2008. Despite being significantly more effective than standard chemotherapies at inducing apoptosis in pancreatic cancer cells, the pleiotropic effects of proteasome inhibition have also been shown to include the induction of several antiapoptotic mechanisms in various cell types. These include downregulation of proapoptotic JNK activity through induction of MAP kinase phosphatase-1 Shi et al., 2006 ; decreased caspase-8 activation through cFLIP induction Liu et al., 2007 and increased heat shock protein expression McConkey and Zhu, 2008. Likewise, we have recently shown that proteasome inhibition is able to increase EGFR, ERK, AKT and JNK signaling in pancreatic cancer cell lines and that these signals are predominantly antiapoptotic Sloss et al., 2008. TopResults and discussion

In this study we sought to determine the biochemistry behind EGFR activation in response to proteasome inhibition. We first confirmed that treatment with the second-generation proteasome inhibitor NPI-0052 resulted in EGFR activation using various PDA cell lines: BxPC3, Capan2 and Panc1. These cell lines account for the various genetic lesions found in PDA including KRas activation and/or loss of one or more of the tumor suppressors p53, p16INK4A and SMAD4. NPI-0052 treatment resulted in a time-dependent activation of EGFR, as measured by detection of tyrosine-1173 phosphorylation using immunoblotting analysis Figure 1a. Several other phosphotyrosine residues responsible for downstream EGFR signaling Y1068 and Y1086 were also phosphorylated in response to NPI-0052 treatment Figure 1a and the activation of EGFR by NPI-0052 was also dose dependent Figure 1b. We similarly determined whether other HER family members were also activated and found that NPI-0052 treatment did not activate either HER2 or HER3 and that HER4 expression was not detectable in any of the cell lines tested data not shown. The proteasome activity of all three cell lines showed low nano- or picomolar sensitivity to NPI-0052 treatment, which correlated with the cells sensitivity to NPI-0052-induced death Supplementary Figure 1. Increases in proteasome inhibitor-induced EGFR activation have been reported in squamous cell carcinoma Lorch et al., 2007, whereas in breast cancer cell lines bortezomib treatment reduced the activity of EGFR Codony-Servat et al., 2006. These studies suggest that the response of EGFR to proteasome inhibition is cell-type specific.Figure 1.

NPI-0052 induces EGFR activation in a time- and dose-dependent manner and induces HER family ligand expression. BxPC3, Panc1, Capan2 were obtained from the ATCC Rockville, MD, USA. Capan2 and Panc1 cells were grown in Dulbecco s modified Eagle s medium DMEM ; BxPC3 in RPMI. Media were supplemented with 10 fetal bovine serum and 100μg/ml penicillin and streptomycin. a BxPc3, Panc1 and Capan2 cells were treated with 100nM NPI-0052 Nereus Pharmaceticals, San Diego, CA, USA for the indicated times or b with the indicated concentrations of NPI-0052 for 4h. Whole-cell extracts were prepared in radioimmunoprecipitation assay RIPA buffer 50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM EDTA, 1 Triton X-100, 1 sodium deoxycholate, 0.1 s.d., 1μg/ml aprotinin, 1μg/ml leupeptin, 1μg/ml pepstatin, 1mM Na3VO4, 50mM b-glycerophosphate, 1mM PMSF and immunoblotted for the indicated pTyr-residues on EGFR or the total form of EGFR. c BxPc3 cells were treated with 100nM NPI-0052 for the indicated times. Total RNA was isolated using the RNeasy mini kit Qiagen, Valencia, CA, USA and 250ng of RNA was used to measure the relative expression of EGFR ligands by RT–qPCR analysis using the indicated hydrolysis probes purchased from Applied Biosystems Foster City, CA, USA in 50μl reactions performed using One Step qRT–PCR Invitrogen, Carlsbad, CA, USA on an ABI7300 system. Glyceraldehyde-3-phosphate dehydrogenase GAPDH was used as control. Error bars represent standard error of the mean s.e.m. of at least three separate experiments, Pd BxPc3 cells were treated with 100nM NPI-0052 for the indicated times and expression of the indicated proteins was measured by immunoblotting.Full figure and legend 228K

Overexpression of many of the HER family ligands including EGF, HB-EGF, amphiregulin and epiregulin has been shown to be involved in pancreatic cancer cell growth, metastasis and to correlate with poor prognosis Zhu et al., 2000; Hansel et al., 2003; Wang et al., 2007b. We proceeded to analyze the expression of five EGFR ligands in response to proteasome inhibitor treatment. RT–qPCR analysis showed a time-dependent increase in the mRNA level of EGF, HB-EGF and epiregulin with significant increases at 4, 8 and 24h after treatment. There was no significant change in the expression of amphiregulin or transforming growth factor-α Figure 1c. Cellular HB-EGF protein levels were maximal after 8h NPI-0052 treatment, which corresponded to the peak in EGFR activation Figure 1d. Interestingly the EGF protein levels did not correlate with the activation of EGFR Figure 1d nor the increase in EGF mRNA Figure 1c. Epiregulin protein was undetectable by immunoblotting in treated or untreated cells data not shown. To determine if the mRNA increases were a result of mRNA stabilization or the induction of de novo gene expression, we used the transcriptional inhibitor actinomycin-D. Actinomycin-D prevented NPI-0052-induced increases in both EGF and HB-EGF mRNA Figure 2a and abrogated the increase of HB-EGF protein Figure 2b indicating the response is a transcriptional event. As HB-EGF functions both as a juxtacrine, membrane-bound protein and as an extracellular ligand, we used enzyme-linked immunosorbent assay to determine the levels of both cellular HB-EGF and the cleaved s-HB-EGF. NPI-0052 treatment significantly increased both cellular and cleaved s-HB-EGF levels Figure 2c. Neutralizing antibodies were then used to sequester shed ligands from the media and as shown in Figure 2d, anti-HB-EGF, but not anti-EGF or anti-EREG, antibodies were able to reduce NPI-0052-induced EGFR activation. Antibody controls showing abrogation of ligand-induced EGFR activation by all three neutralizing antibodies are shown in Supplementary Figure 2b. Interestingly, the increases in HB-EGF shedding into the media were similar to the results previously reported for increases in HB-EGF shedding in response to the chemotherapy treatments SN-38 and doxorubicin Wang et al., 2007a. It remains to be determined if a common stress response mechanism underlies these similar findings.Figure 2.

Ligand expression is actinomycin-D sensitive. BxPc3 cells were treated with vehicle or 100nM NPI-0052 in the presence or absence of 5μg/ml ActinomycinD Sigma, St Louis, MO, USA for 4h. The relative expressions of HB-EGF and EGF were measured by a RT–qPCR or b immunoblotting. NPI-0052 treatment induces HB-EGF shedding. c BxPc3 cells were treated for 8h with vehicle or 100nM NPI-0052, growth media and whole-cell extracts were analyzed for levels of HB-EGF expression by enzyme-linked immunosorbent assay ELISA using antibodies purchased from R D Systems Minneapolis, MN, USA. Samples were precleared by centrifugation at 5000g for 5min at 4 C and 100μl per well used for analysis as described in the manufacturer s protocol and as described previously Wang et al., 2007a. A standard curve was generated using rh-HB-EGF R D Systems and used to quantify unknowns. Error bars represent s.e.m. of at least three separate experiments. Pt-test. d BxPc3 cells were treated for 8h with vehicle or 100nM NPI-0052 in the presence of the indicated amounts of neutralizing antibodies R D Systems. Lysates were analyzed for activation of EGFR by immunoblot. Histogram represents mean quantification by scanning densitometry of at least three separate immunoblot bands representative blots are shown in Supplementary Figure 2a. Error bars represent s.e.m. Pμg/ml of neutralizing antibody.Full figure and legend 128K

All EGFR ligands are cleaved at the cell surface by MMP/ADAMs to release the active ligand. To confirm if ectodomain shedding was involved in the activation of EGFR by proteasome inhibition, we treated pancreatic cancer cells with NPI-0052 in the presence of the broad-spectrum MMP/ADAM inhibitor GM6001. GM6001 was able to prevent NPI-0052-induced activation of EGFR in a dose-dependent manner Figure 3a and prevent cleavage and release of s-HB-EGF into the media Supplementary Figure 2c. Crm-197 a potent and specific inhibitor of human HB-EGF also completely abrogated the activation of EGFR by NPI-0052 Figure 3b.

Activation of EGFR is MMP/ADAM and HB-EGF dependent. BxPc3 cells were treated for 8h with vehicle or 100nM NPI-0052 with either a the indicated concentrations of the MMP/ADAM inhibitor GM6001 Sigma or b the indicated concentrations of the HB-EGF inhibitor crm-197 EMD Biosciences, Madison, WI, USA and expression of the indicated proteins was measured by immunoblotting. Knockdown of HB-EGF but not EGF or epiregulin prevents EGFR activation. Control Plko1 vector, EGF NM_001963.2-2240s1c1 ; HB-EGF NM_001945.1-827s1c1 or EREG NM_001432.1-341s1c1 Sigma were used to produce lentiviral particles in 293 T cells as described in the manufacturer s protocol. After lentiviral transduction, stable BxPC3 clones were generated by passaging cells in 5μg/ml Puromycin Fisher, Waltham, MA, USA. Control cells Bx-Plko1 or cells lacking HB-EGF Bx-shHB, EGF Bx-shEGF or epiregulin Bx-shEREG were treated with vehicle or 100nM NPI-0052 for 8h and c the relative expression of ligands were measured using RT–qPCR error bars represent s.e.m., P-values represent analysis by Student s t-test or d cell lysates were immunoblotted for the indicated proteins.Full figure and legend 205K

Together these data suggest that HB-EGF expression and cleavage are responsible for proteasome inhibitor-induced EGFR activation. Similar data from other groups have implicated HB-EGF and its cleavage by MMP/ADAMs in the activation of EGFR in response to several types of stimuli including growth factors Reznik et al., 2008, GPCR activation Miyamoto et al., 2006 and cellular stresses including chemotherapeutics and inflammatory cytokines Takenobu et al., 2003; Wang et al., 2007a. We additionally assessed if the lack of NPI-0052-induced EGFR activity in the breast cancer cell line SKBR3 data not shown; Codony-Servat et al., 2006 was due to a lack of HB-EGF induction. When treated with NPI-0052 both BxPc3 and SKBR3 cells showed marked increases in HB-EGF mRNA levels Supplementary Figure 3a whereas only BxPc3 cells had detectable HB-EGF protein expression Supplementary Figure 3b. This would suggest that the absence of proteasome-induced EGFR activity in SKBR3 cells is due to a lack of HB-EGF protein translation. A recent study has shed light on a possible explanation for this discordance between transcription and translation in response to proteasome inhibition. Zhu et al. 2009 have shown the phosphorylation state of eIF2α and disruption of polysome complexes can account for transcriptional independent suppression of protein translation in response to proteasome inhibitors. The authors suggest that proteasome inhibitors can specifically suppress translation of some proteins more than others and that the abundance of proteins, which are degraded by non-proteasome-dependent means, will be decreased in response to proteasome inhibitor treatment.

To confirm that HB-EGF is required for the activation of EGFR in response to NPI-0052, we engineered stable clones of BxPc3 cells lacking HB-EGF Bx-shHB, EGF Bx-shEGF or epiregulin Bx-shEREG using lentiviral-mediated shRNA transduction. Abrogation of ligand expression was confirmed by RT–qPCR and activation of EGFR was assessed by immunoblotting. Despite effective knockdown of all three ligands Figure 3c only cells lacking HB-EGF showed a significant reduction in the activation of EGFR in response to NPI-0052 Figure 3d. These data confirm that HB-EGF and not EGF or epiregulin is required for EGFR activation in response to proteasome inhibitor treatment. Knockdown of HB-EGF also sensitized BxPc3 cells to NPI-0052 treatment, shifting the Ic50 from 154nM for Bx-Plko1 cells to 50nM for Bx-shHB cells data not shown.

We next sought to identify the signaling pathways involved in the induction of HB-EGF expression. Previous work by Ingram et al 2003 has shown that reactive oxygen species ROS, ERK and p38-MAPK pathways are involved in HB-EGF expression in response to stress stimuli. Our study results show that inhibition of p38-MAPK by SB203580 and ROS by N-acetyl-cysteine, NAC prevented NPI-0052-induced expression of HB-EGF and activation of EGFR, whereas inhibition of ERK by PD98059 did not Figure 4a. p38-MAPK and ROS inhibition were also able to significantly reduce HB-EGF transcription Figure 4b. To ascertain if ROS was upstream of p38-MAPK activation, we treated BxPc3 cells with increasing concentrations of NAC Figure 4c. NPI-0052-induced p38-MAPK activity decreased in an NAC dose-dependent manner, concomitantly with decreases in both HB-EGF and active EGFR levels. These results are in agreement with a previous study that highlighted the need for p38-MAPK and HB-EGF in the activation of EGFR in response to both osmotic and oxidative stresses Fischer et al., 2004. Figure 4.

HB-EGF induction and EGFR activation is dependent on reactive oxygen species ROS and p38-MAPK but not ERK. a BxPc3 cells were treated for 8h with vehicle or 100nM NPI-0052 with inhibitors of the ERK pathway 10μM PD98059 ; the p38 MAPK pathway 2.5μM SB203580 or ROS 10mM N-acetyl-cysteine NAC. Cell lysates were analyzed for the indicated proteins by immunoblot or b for HB-EGF mRNA levels by RT–qPCR. Error bars represent s.e.m. of at least two experiments. Pt-test vs NPI-treated control. c BxPc3 cells were treated for 8h with vehicle or 100nM NPI-0052 with the indicated concentrations of NAC. Cell lysates were analyzed for the indicated proteins by immunoblot. d Panc1 cells in six-well plates were transfected with 50nM of either scrambled or anti-SP-1 stealth siRNA Invitrogen, using 5μl of RNAiMax Invitrogen, in 500μl of Optimem Invitrogen, added to a final volume of 3ml of antibiotic free media per well as described in the manufacturer s protocol. After 48h cell lysates were analyzed for relative HB-EGF mRNA levels by RT–qPCR or by immunoblot for the indicated proteins. Error bars represent s.e.m. of at least three experiments. Pt-test.Full figure and legend 149K

We subsequently sought to identify the transcription factors downstream of the ROS/p38-MAPK pathway. Previous work has shown that p38-MAPK is able to phosphorylate and activate the transcription factor SP-1 D Addario et al., 2006 and that the HB-EGF promoter contains several SP-1-binding sites Edwards et al., 2009. To assess if SP-1 was involved in the induction of HB-EGF in response to NPI-0052, we transfected Panc1 cells with siRNA targeted against SP-1. At 48h after transfection, SP-1 mRNA levels decreased to less than 7 of basal Supplemental Figure 4. Knockdown of SP-1 mRNA resulted in a significant decrease in both the mRNA and protein level of HB-EGF and a corresponding decrease in the NPI-0052-induced activation of EGFR Figure 4d. This suggests the transcription factor SP-1 is important in the induction of HB-EGF in response to proteasome inhibition.

Although we have shown here that HB-EGF is required for the activation of EGFR in tumor epithelial cells, it is also likely that increased HB-EGF expression and shedding in response to proteasome inhibitor treatment may additionally induce paracrine effects within the tumor microenvironment. This hypothesis is supported by recent work that has shown HB-EGF expression and EGFR signaling are responsible for pancreatic fibrosis Means et al., 2003; Blaine et al., 2009 ; and that extracellular matrix deposition by the stroma can increase pancreatic cancer chemoresistance Miyamoto et al., 2004. HB-EGF and EGFR signaling in pancreatic tumor–stroma interactions may also explain the discordance between the limited efficacy of EGFR inhibitors in pancreatic cancer cell culture systems and the positive effects of EGFR inhibitors in murine models of pancreatic cancer and in the clinic Buck et al., 2006; Moore et al., 2007; Sloss et al., 2008. Moreover, as the dense stroma of pancreatic tumors has been implicated in the poor vascularization of tumors Olive et al., 2009. HB-EGF inhibition could potentially reduce stromal growth and increase drug penetration of pancreatic tumors. When the results shown here are viewed in this context, it is clear that HB-EGF targeting therapies will have multiple antipancreatic tumor effects: including reduced EGFR signaling in pancreatic cancer epithelial cells and reduced stromal growth and desmoplastic reaction. These combined effects should lead to a decrease in the growth and invasion of pancreatic tumors and an increase in chemosensitivity.

In summary, we have previously shown that proteasome inhibition can activate EGFR in pancreatic cancer cells and that inhibition of EGFR increases the efficacy of proteasome inhibitor treatment in a mouse xenograft model of pancreatic cancer Sloss et al., 2008. In this study we examined the role of EGFR ligands in the activation of EGFR in response to proteasome inhibition. Our study results indicate that proteasome inhibitor treatment induced the expression of EGF, epiregulin and HB-EGF but that only HB-EGF knockdown could abrogate EGFR activation. Activation of EGFR was also sensitive to both MMP/ADAM inhibition and ligand sequestration by an anti-HB-EGF antibody. These data indicate that HB-EGF expression and ligand cleavage are required for EGFR activation in response to proteasome inhibition. In addition, we found that induction of HB-EGF expression by NPI-0052 requires ROS, subsequent downstream p38-MAPK signaling and involves the transcription factor SP-1. Taking into consideration the significant antitumor potential of proteasome inhibition in pancreatic cancer models and the antiapoptotic effects of HB-EGF through both autocrine and paracrine mechanisms, we believe the combination of proteasome inhibitors and HB-EGF inhibitors warrant further investigation at both the preclinical and clinical levels.

Activation of the Epidermal Growth Factor Receptor in EGFR activation and inhibition of Colonic epithelial cells were isolated using a modified protocol.

Lung Cancer Mutation Panel EGFR, KRAS, ALK Although EGFR TKIs can block EGFR activation, they cannot block the activity of the mutated KRAS protein.

Ligand activation of the epidermal growth factor receptor EGFR leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be.

Epidermal growth factor receptor nucleotide exchange factor Sos 1, and, together, they regulate the EGFR mediated activation standard protocol.

Activation of EGFR by proteasome inhibition requires HB-EGF in in the manufacturer s protocol and epidermal growth factor receptor activation by.

TYROSINE KINASE SERIES: EGFR L858R EGFR Activation of EGFR triggers activation of the ERK Protocol Dilute enzyme.