The MEK/ERK Pathway Promotes NOTCH Signalling in Pancreatic Cancer Cells

<div><p>Activation of the NOTCH receptors relies on their intracellular proteolysis by the gamma-secretase complex. This cleavage liberates the NOTCH intracellular domain (NIC) thereby allowing the translocation of NIC towards the nucleus to assemble into a transcriptional platform. Little information is available regarding the regulatory steps operating on NIC following its release from the transmembrane receptor up to its association with transcriptional partners. Interfering with these regulatory steps might potentially influences the nuclear outcome of NOTCH signalling. Herein, we exploited a reliable model to study the molecular events occurring subsequent to NOTCH1 cleavage. In pancreatic cancer cells, pulse of NOTCH1 activation led to increased expression of NOTCH target genes namely HES1 and c-MYC. We uncovered that, upon its release, the NOTCH1 intracellular domain, NIC1, undergoes a series of post-translational modifications that include phosphorylation. Most interestingly, we found that activation of the MEK/ERK pathway promotes HES1 expression. Inhibition of the gamma-secretase complex prevented the MEK/ERK-induced HES1 expression suggesting a NOTCH-dependent mechanism. Finally, higher levels of NIC1 were found associated with its transcriptional partners [CBF1, Su(H) and LAG-1] (CSL) and MASTERMIND-LIKE 1 (MAML1) upon MEK/ERK activation providing a potential mechanism whereby the MEK/ERK pathway promotes expression of NOTCH target genes. For the first time, our data exposed a signalling pathway, namely the MEK/ERK pathway that positively impacts on NOTCH nuclear outcome. </p> </div

Activation of the MEK/ERK pathway promotes NOTCH signalling.

<p><b>A</b>. MIA PaCa-2 cells were pre-treated (+) or not (-) with DAPT (25μM) overnight. Cells were then treated with PMA (100nM) for the indicated time periods. <b>B</b>. MIA PaCa-2 cells were left untreated (-) or treated with EGTA (4mM) for 15min (+). EGTA-containing medium was removed and replaced by normal culture media (Ca²⁺) containing DMSO, PMA (100nM) or PMA + U0126 (10μM) for the indicated time periods. <b>A</b>. and B. Cells were lysed and Western blot analyses were performed using the indicated antibodies. <b>C</b>. From similar experiments presented in B., a graphic representation depicting relative HES1 expression, for each time period, in treated cells (PMA, PMA+U0126) compared to control cells (DMSO-treated) is shown. <b>D</b>. From similar experiments presented in B., a graphic representation depicting relative c-MYC expression, for each time period, in treated cells (PMA, PMA+U0126) compared to control cells (DMSO-treated) is shown. <b>C</b>. and D. The results are the means ± SEM of at least three independent experiments. * p<0.05, ** p<0.005, ***p<0.0005 compared to the corresponding time period in DMSO-treated cells. # p<0.05, ## p<0.005, ### p<0.0005 compared to the corresponding time period in PMA-treated cells. </p

Activation of the MEK/ERK pathway promotes NIC1 association with its transcriptional partners.

<p>MIA PaCa-2 cells were left untreated (-) or treated with EGTA (4mM) for 15min (+). EGTA-containing medium was removed and replaced by normal culture media (Ca²⁺) containing DMSO, PMA (100nM) or PMA + U0126 (10μM) for 90min. A. NIC1, MAML1 and CSL expression levels were assessed by western blot using the appropriate antibodies. <b>B</b>. CSL and MAML1 were immunoprecipitated (IP) prior to western blot analyses using the indicated antibodies. <b>C</b>. CSL was immunoprecipitated (IP) followed by western blot analyses of NIC1 (left). A graphic representation depicting the relative amount of NIC1 co-immunoprecipitated with CSL (NIC1/CSL) is shown (right). <b>D</b>. MAML1 was immunoprecipitated (IP) followed by western blot analyses of NIC1 (left). A graphic representation depicting the relative amount of NIC1 co-immunoprecipitated with MAML1 (NIC1/MAML1) is shown (right). <b>C</b>. and D. The results are the means ± SEM of at least three independent experiments. * p<0.05 compared to DMSO-treated cells. </p

NIC1 is phosphorylated in pancreatic cancer cells.

<p>NIC1 was immunoprecipitated (IP NIC1) from total lysates of exponentially growing MIA PaCa-2 (MIA), MIA PaCa-2 cells treated with EGTA (4mM) for 15min and then returned to their normal culture media for 90min, or exponentially growing BxPC-3 (Bx). Following NIC1 immunoprecipitation, phosphatase assays were performed (+) using the lambda phosphatase (λ phosphatase). The IP NIC1 not incubated with the lambda phosphatase is indicated as pNIC1. A representative Western blot using an antibody against NIC1 is shown.</p

Expression of a constitutive active form of MEK1 induces NIC1 post-translational modifications when expressed in HEK293T cells.

<p><b>A</b>. HEK293T were transfected with pLIA-NIC1 (MYC-tagged) construct together with a cDNA encoding a wild-type (WT) or constitutive active (CA) version of MEK1 (HA-tagged). Cells were lysed 24h post-transfection. NIC1 expression was analysed by western blot using an anti-MYC antibody. The expression level of the exogenous MEK1 was evaluated by western blot using an anti-HA antibody. Dual-phosphorylated and total ERK1/2 expression levels were assessed using specific antibodies. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. <b>B</b>. HEK293T were transfected with pLIA-NIC1 construct together with a cDNA encoding MEK1 WT or MEK1 CA. Cells were lysed and NIC1 was immunoprecipitated (IP NIC1) using an anti-MYC antibody. Phosphatase assays were then performed (+) using the lambda phosphatase (λ phosphatase). The phosphorylated forms of NIC1 are denoted pNIC1. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. NIC1 expression was analysed by western blot using an anti-MYC antibody. <b>C</b>. NIC1 was immunoprecipitated (IP NIC1) from pLIA-NIC1 transfected HEK293T using an anti-MYC antibody. Kinase assays were then performed as described in experimental procedures. The autoradiography depicting phosphorylated NIC1 (pNIC1) is shown. Following electrotransfer of the gel, the amount of NIC1 immunoprecipitated was confirmed by western blot (WB) using an anti-MYC antibody.</p