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Supplementary Figure Legends.
Supplementary Figure 1. Evaluation of correlation of basal levels of EGFR, pEGFR, and
downstream effectors of the RAS-RAF-MEK-ERK pathway and sensitivity to selumetinib in
CRC and NSCLC cell lines. A) Cancer cells were analyzed by immunoblotting for EGFR, pEGFR, MEK 1/2, p-MEK1/2, AKT, p-AKT, RAS Total (RAS-T) levels and RAS activity (RASACT). B) Signal intensity was quantified using the Image J freeware. Cells were grouped as either
sensitive (IC50 at 96 hrs ≤ 1μM) or resistant (IC50 at 96 hrs > 1 μM) to the MEK inhibitor
selumetinib. Relative EGFR, p-EGFR, MEK 1/2, p-MEK1/2, AKT, p-AKT, RAS Total (RAS-T)
levels and RAS activity (RAS-ACT) are shown for the two groups.
Supplementary Figure 2. Gene mutation analysis and sensitivity to selumetinib in NSCLC and
CRC cell lines. Venn diagram show the distribution of KRAS, PI3CA, BRAF and N-RAS gene
mutations.
Supplementary Figure 3. Heat map results from microarray gene expression profiles of
selumetinib-sensitive and -resistant NSCLC and CRC cell lines. A Student's t test with a
Benjamini–Hochberg multiple test correction shows 39 differentially expressed genes in CRC and
NSCLC cell lines according to their sensitivity or resistance to selumetinib.
Supplementary Figure 4. Schematic representation of cAMP-dependent protein kinase (PKA)
involvement in intracellular signaling. Growth factors, such as EGF, upon binding to growth
factor receptors (tyrosine kinase receptors, TKR), activate, via the adapter protein growth factor
receptor-bound protein 2 (GRB2), the guanine nucleotide exchange factor son of sevenless (SOS)
and in turn the small GTP-binding protein, RAS. Recruitment of RAF-1 to the plasma membrane by
RAS leads to its activation triggering the MAPK cascade and cell proliferation. Binding of an
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extracellular ligand to a transmembrane G-protein coupled receptor (GPCR) alters the conformation
of the associated heterotrimeric G protein, causing dissociation of the Gαs and Gβγ subunits and
initiating a cascade of intracellular events. The subunit Gαs activates the adenylatecyclase enzyme
(ADCY7) which converts ATP into cAMP. cAMP activates protein kinase A (PKA) binding to the
regulatory (R) subunits, which causes a conformational change that releases the active catalytic (C)
subunits. The catalytic subunits of PKA phosphorylate proteins at specific Ser or Thr side chains
such as the transcription factors of cAMP response element binding (CREB) family. PKA
represents the main intracellular effector of cAMP signaling. The Catalitic subunity of PKA is then
capable of activates the kinase B-Raf promoting the MAPK cascade. A-kinase anchoring proteins
(AKAPs) binding to PKA can interact with other signaling molecules. Moreover, PKA catalytic
subunit is able to activate the retinoic acid pathway (RA signaling).
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