Review Article

The MAPK Pathway Across Different

Malignancies: A New Perspective
Mauricio Burotto, MD; Victoria L. Chiou, MD1; Jung-Min Lee, MD1; and
Elise C. Kohn, MD1,2

The mitogen-activated protein kinase/extracellular signal-regulated (MAPK/ERK) pathway is activated by upstream genomic events
and/or activation of multiple signaling events in which information coalesces at this important nodal pathway point. This pathway is
tightly regulated under normal conditions by phosphatases and bidirectional communication with other pathways, like the protein ki-
nase B/mammalian target of rapamycin (AKT/m-TOR) pathway. Recent evidence indicates that the MAPK/ERK signaling node can
function as a tumor suppressor as well as the more common pro-oncogenic signal. The effect that predominates depends on the in-
tensity of the signal and the context or tissue in which the signal is aberrantly activated. Genomic profiling of tumors has revealed
common mutations in MAPK/ERK pathway components, such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF). Currently
approved for the treatment of melanoma, inhibitors of BRAF kinase are being studied alone and in combination with inhibitors of the
MAPK and other pathways to optimize the treatment of many tumor types. Therapies targeted toward MAPK/ERK components have
various response rates when used in different solid tumors, such as colorectal cancer and ovarian cancer. Understanding the differen-
tial nature of activation of the MAPK/ERK pathway in each tumor type is critical in developing single and combination regimens,
because different tumors have unique mechanisms of primary and secondary signaling and subsequent sensitivity to drugs. Cancer
2014;000:000-000. V C 2014 American Cancer Society.

KEYWORDS: mitogen-activated protein kinase (MAPK), v-raf murine sarcoma viral oncogene homolog (BRAF), extracellular signal-
regulated kinase (ERK), mitogen-activated protein kinase-kinase (MEK), signaling, melanoma, ovarian cancer, colorectal cancer.

INTRODUCTION
Great advances have been made toward understanding the genomic characterization of solid and hematologic malignan-
cies. Collaborative projects, such as The Cancer Genome Atlas (TCGA),1 have revealed the molecular complexity of
tumors and also the challenges inherent in data interpretation and clinical application. Differentiation of targetable driver
mutations from genetic and genomic noise and passenger mutations is 1 of the most important goals of genome and epige-
nome analysis.2,3 Driver mutations lead to dysregulation of signaling pathways, increasing malignant behavior.4 It has
been demonstrated that many of these gain-of-function driver mutations are druggable, leading to the development of
small molecules and antibodies that target specific events, such as the ligand-binding site of the receptor or the adenosine
triphosphate (ATP) binding site in the kinase domain of specific kinase proteins.
The mitogen-activated protein kinase (MAPK) cascade is a critical pathway for human cancer cell survival, dissemi-
nation, and resistance to drug therapy.5 The MAPK/extracellular signal-regulated kinase (ERK) pathway is a convergent
signaling node that receives input from numerous stimuli, including internal metabolic stress and DNA damage pathways
and altered protein concentrations, as well as through signaling from external growth factors, cell-matrix interactions, and
communication from other cells.6 Mutated genes responsible for the regulation of cell fate, genome integrity, and survival
can lead to increased protein amplification and alter the tumor microenvironment, thus overactivating the pathway.7
These mutations can occur upstream in membrane receptor genes, such as epithelial growth factor receptor (EGFR),8 in
signal transducers (RAS),9 regulatory partners (Sprouty),10 and downstream kinases that belong to the MAPK/ERK path-
way itself (BRAF) (Fig. 1).11,12 Several mutations involving the MAPK/ERK pathway have been identified in human can-
cers and are ripe for targeting. Current and future drug-development efforts will need to alter and regulate tumor
signaling in this complex network of codependent pathways.

Corresponding author: Mauricio Burotto, MD, Building 10, Room 12N226, 9000 Rockville Pike, Bethesda, MD 20892; Fax: (301) 451-0456;
mauricio.burottopichun@nih.gov
1
Women’s Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland; 2Cancer Therapy Evaluation Program, Division of
Cancer Treatment and Diagnostics, National Cancer Institute, Bethesda, Maryland

DOI: 10.1002/cncr.28864, Received: February 13, 2014; Revised: April 22, 2014; Accepted: May 8, 2014, Published online Month 00, 2014 in Wiley Online
Library (wileyonlinelibrary.com)

Cancer Month 00, 2014 1

Review Article
The MAPK Pathway and its Regulation
There are 4 independent MAPK pathways composed of 4
signaling families: the MAPK/ERK family or classical
pathway and the Big MAP kinase-1 (BMK-1), c-Jun N-
terminal kinase (JNK), and p38 signaling families.13
These families share a basic organization composed of 2
serine/threonine kinases and 1 double-specificity threo-
nine/tyrosine kinase.14 Generically, these kinases are des-
ignated from upstream to downstream, closer to the
nucleus, as MAPK kinase-kinase (MAPKKK), MAPK ki-
nase (MAPKK) and MAPK (Fig. 1).5 The canonical
MAPK/ERK pathway is composed of 3 types of
MAPKKK: A-RAF, B-RAF, and RAF-1 or C-RAF ki-
nases. BRAF is the gene most commonly mutated at this
level in human cancer. One level below are the MAPKKs,
which are composed of MEK1 and MEK2. Finally, fur-
ther downstream are ERK1 and ERK2, which are the final
effectors of the MAPK pathway.15
ERK phosphorylation results in the activation of
multiple substrates that are responsible for the stimulation
of cell proliferation. Spatial localization of ERK deter-
mines target substrates and later effects within the cell.6
When located in the cytoplasm, ERK phosphorylates
cytoskeletal proteins that affect cell movement and traf-
ficking,16 metabolism, cell adhesion, and nodal regulation
of other pathways.17 Cytoplasmic substrates include ribo-
somal S6 kinases (RSKs), which regulate the glycogen syn-
thase kinase 3 (GSK3) involved in metabolism, and L1
adhesion molecule, a protein of neural origin that partici-
pates in cell adhesion.18,19 Minutes after MAPK/ERK
activation, ERK detaches from cytoplasmic anchoring
proteins and translocates to the nucleus to exert its tran-
scriptional regulation.20 Active ERK in the nucleus causes
phosphorylation and activation of various transcription
factors, such as carbamoyl phosphate synthetase II (CPS
II) linking with synthesis of DNA or p90RSK and pro-
moting cell cycle progression. These 2 events are integral
in MEK/ERK stimulation of cell proliferation.21,22 In
immune cells, activated ERK is also a component of the
innate response in different steps of the inflammatory cas-
cade, increasing the expression of tumor necrosis factor
alpha (TNF-a) and inducible nitric oxide synthase
(iNOS).23
In addition to spatial activation, the final effect of
the MAPK/ERK pathway is modulated by timing, dura-
tion, and intensity of its signal. Winters et al examined the
MAPK/ERK cascade at different times points in colo-
rectal cancer (CRC) cell lines under the combination of
carboxyamido-triazole, an intracellular calcium regulator,
plus the selective cyclooxygenase 2 inhibitor celecoxib.
2 Cancer
Suppression of ERK activation occurred in the first hour
of treatment, in contrast to the sustained ERK phospho-
rylation after 9 days of treatment.24 Indeed, cells interpret
and respond differently to small changes in the levels of
MAPK/ERK activation. As described by Murphy et al, c-
FOS, an early gene product of MAPK/ERK activation,
works as a sensor of the duration of ERK stimulation.
When the MAPK/ERK signal is transient, c-FOS is unsta-
ble and degraded in the nucleus; but, if the signal is sus-
tained, then c-FOS is phosphorylated, and specific
domains are exposed, promoting more ERK activation.25
The procarcinogenic or proapoptotic signaling of this
pathway depends on the timing and duration of MAPK/
ERK activation.
Specific proteins, such as kinase suppressor Ras-1
(KSR1), work as the main scaffold for proteins related to
MAPK/ERK pathway activation. Cytoplasmic proteins,
Sprouty and Spred, directly inhibit the pathway26 by
removing activating phosphate groups from ERK, thereby
decreasing its ability to phosphorylate its substrates.12
Thus, there are regulatory events in both the cytoplasm
and the nucleus, along with spatial and temporal regula-
tion, that fine tune the output of the MAPK/ERK
pathway.
Overactivated and Oncogenic Drivers of the
MAPK Pathway as Therapeutic Targets
Cellular proliferation is driven by an intricate network of
regulated, interdependent signals. The complexity of the
MAPK pathway is not random; it allows for the periodic
environmental adaptation necessary for activation and
regulation of the coordinated events critical for cell sur-
vival.27 MAPK/ERK pathway activation and subsequent
interactions are highly regulated processes that are deregu-
lated in cancer cells. Stimulation of growth factor recep-
tors in the cell membrane leads to activation of 2 different
but interconnected, pivotal pathways: the phosphoinosi-
tide 3-kinase (PI3K) signal, which causes activation of
protein kinase B (AKT) and its downstream substrates,
and the MAPK/ERK pathway (Fig. 1). Both drive cell
proliferation, survival, and dissemination. The PI3K/
AKT pathway also promotes anabolism; whereas the
MAPK/ERK pathway is more active in proliferation and
invasion.5 Up-regulation of MAPK/ERK signaling occurs
as a result of overexpression or aberrant activation of re-
ceptor tyrosine kinases (RTKs) or their immediate down-
stream targets: PI3K, proto-oncogene tyrosine-protein
kinase Src (SRC), and RAS.
Normal MAPK/ERK function is also responsible for
tumor suppression through the induction of senescence
Month 00, 2014
 Tissue-Specific MAPK Signaling/Burotto et al

Figure 1. This is a model of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Af-
ter membrane receptor activation, adaptor proteins recruit RAS proteins to activate steps that conclude with ERK activation.
Successive steps of phosphorylation amplify the signal, Raf!MAPK kinase (MEK)!ERK, until ERK activates its cytoplasmic and/
or nuclear targets. Regulatory phosphatases, Sprouty and Spred, modulate the intensity of the signal. The phosphatidylinositol-3-
kinase/protein kinase B (PI3K/AKT) pathway interacts with the MAPK/ERK node under normal conditions and in the cancer cell.
Target cytoplasmic proteins include ribosomal S6 kinases (RSKs), glycogen synthase kinase 3 (GSK3), and adhesion molecule L1
(LI). Additional proteins in the nucleus include copalyl diphosphate synthase 2 (CPS2) and 90-kDa ribosomal protein S6 kinase
(p90Rsk). EGFR indicates epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor; VEGFR, vascular
endothelial growth factor receptor; RTK, receptor tyrosine kinase; SRC, proto-oncogene tyrosine-protein kinase Src; K-Ras, v-Ki-
ras Kirsten rat sarcoma viral oncogene homolog; MAPKKK, MAPK kinase-kinase; A-Raf, serine/threonine-protein kinase A-Raf;
MAPKK, MAPK kinase; mTOR, mammalian target of rapamycin.

and the ubiquitinization and degradation of proteins nec-
essary for cell cycle activity and survival.28 Senescence
involves the inhibition of cell proliferation through termi-
nal cell cycle arrest.29 Abnormal activation of MAPK/
ERK by RAS causes degradation of proteins required for
both migration and progression through the cell cycle, as
demonstrated in a model of normal fibroblasts and vali-
dated in benign prostate tumors. In those tumors, high
levels of phosphorylated (phospho)-ERK were coex-
pressed with markers of senescent p16INK4a and promy-
elocytic leukemia protein (PML), a marker of protein
degradation.28 In addition, a screening study using a panel
of silencing RNAs (shRNAs) against MEK1 increased
lymphoma genesis in MYC-expressing lymphoid cells,
demonstrating that MEK1 has tumor suppressor proper-
ties and that the function of MEK1 kinase is context-
dependent.30
Genetic mutations can dysregulate kinase activity
and hyperactivate the MAPK pathway during induction
and progression of tumorigenesis. Many oncogenic driver
mutations have been identified in genes upstream of
MAPK/ERK, varying across cancer types, as indicated in
Table 1. These may include exon 21 mutations in EGFR
or del19EGFR, mutations in v-Ki-ras Kirsten rat sarcoma
viral oncogene homolog (KRAS), and the classic valine to
glutamic acid (V600E) BRAF mutation. These mutated
genes lead to downstream overactivation of the MAPK/
ERK pathway. In general, mutations affecting the

Cancer Month 00, 2014 3

Review Article

TABLE 1. Frequency of Mutations in the Activators and Components of Mitogen-Activated Protein Kinase/
Extracellular Signal-Regulated Kinase Pathway Across Different Tumors

Frequency of Mutations, % (References)

Tumor RAS BRAF MEK ERK

Melanoma 15-29 (Colombino 2012,31 50-60 (Davies 200233) 3-8 (Murugan 2009,34 NR
Edlundh-Rose 200632) Nikolaev 201235)
NSCLC 12-30 (Seo 201236) 37
4 (Cardarella 2013 ) NR NR
Colorectal 34.1 (Cancer Genome Atlas 5-20 (Davies 2002,33 Tol 200939) <3 (Murugan 200934) NR
Network 201238)
Ovarian
HGSOC 0-12 (Sieben 200440) NR NR NR
LGSOC 27-36 (Bell 2005,41 Singer 200342) 33-50 (Bell 2005,41 Singer 200342)
Thyroid 9-27 (Bansal 201343) 45-69 (Xing 2013,44 Cohen 200345) NR NR
Hairy cell NR 79-100 (Xi 2012,46 Tiacci 201147) NR NR

Abbreviations: BRAF, v-raf murine sarcoma viral oncogene homolog; ERK, extracellular signal-regulated kinase; HGSOC, high-grade serous ovarian cancer;
LGSOC, low-grade serous ovarian cancer; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; NR, not reported; NSCLC,
nonsmall cell lung cancer; RAS, v-ras viral oncogene homolog.

MAPK/ERK pathway are singular, independent events.
Infrequently, 2 mutations can be identified in the RAS/
RAF/MEK/ERK pathway within the same tumor, dem-
onstrating tumoral molecular heterogeneity.48 The sensi-
tivity of mutation detection depends on the dominant
population of cells represented in the tumor sample that is
tested and, thus, may not be illustrative of the tumor as a
whole.49
Discovery of specific oncogene mutations that acti-
vate the MAPK pathway has spurred the development of
targeted therapies that apply to multiple tumor types.
Studies in tumor cells with mutant V600EBRAF have
demonstrated that RAF kinase inhibitors prevent ERK
signaling.50 The selective MEK inhibitor PD0325901
decreased cyclin D1 protein expression, thereby decreas-
ing cell proliferation in BRAF-mutant melanoma xeno-
graft models.51 High plasma concentrations of the RAF
inhibitor vemurafenib are associated with strong ERK
pathway inhibition. Patients with advanced-stage,
V600EBRAF-mutated, metastatic melanoma receiving
vemurafenib treatment who achieved >80% inhibition of
cytoplasmic ERK phosphorylation, as observed in paired
pretreatment and on-treatment biopsy samples, demon-
strated clinical evidence of partial remission.52 Recently,
the immunomodulatory effects of BRAF inhibition were
examined, and it was demonstrated that these effects
explained part of the efficacy of vemurafenib in mela-
noma. BRAF and MEK inhibition increased the expres-
sion of melanoma antigens in melanoma cell lines. This
could increase T-cell recognition of the tumor, leading to
a successful immunotherapeutic approach.53
The next proteins downstream, MEK1 and MEK2,
have now been successfully targeted. Selumetinib
exhibited some activity in metastatic biliary cancers in a
study of 28 patients, yielding a response rate of 12% and a
progression-free survival of 3.7 months. Only 2 patients
had RAS mutations, and neither responded to therapy.54
Selumetinib was also studied in 24 patients who had met-
astatic papillary or poorly differentiated thyroid cancer
that was refractory to radioiodine treatment. Selumetinib
increased the iodine-124 uptake in 12 patients, and 5 of 8
patients had responses to radioiodine. Mutations were
detected in 7 of the 8 patient treated; responses were
reported in 4 patients who had neuroblastoma RAS viral
oncogene homolog (NRAS) mutations and in 1 patient
who had a BRAF mutation.55 Docetaxel with or without
selumetinib was studied in a randomized phase 2 trial of
87 patients with metastatic non-small cell lung cancer
who had KRAS mutations. The response rate was 37% in
the experimental arm versus 0% in the arm without selu-
metinib (P<.0001), and the progression-free survival was
5.3 months versus 2.1 months, respectively (P5.014).56
Currently, many ongoing phase 2 clinical trials are explor-
ing the use of agents targeting BRAF (Table 2) and MEK
kinases (Table 3). Many of those trials apply mutational
analyses for study eligibility to enrich for patients who
may be most likely to benefit. Data suggest that this path-
way behavior is not consistent in all settings, which makes
targeting the addictive oncogenic pathway a challenge
across different tumor types.
The MAPK/ERK pathway is a double-edged sword.
Generally, therapeutic inhibition of elements within this
pathway has yielded some benefit. However, small mole-
cule inhibitor therapy aimed at specific protein targets
within the MAPK/ERK pathway has resulted in the devel-
opment of secondary malignancies. RAF inhibitors, as a

4 Cancer Month 00, 2014

 Tissue-Specific MAPK Signaling/Burotto et al

TABLE 2. Phase 2 Ongoing Clinical Trials for BRAF MAPK/ERK pathway (Fig. 2). Thus, the relative impor-
Inhibitors in Patients With Solid Tumors tance of MAPK/ERK in cancer cells depends on the cell
Clinical Trial and/or tissue of origin, the magnitude of addictive de-
pendence on the pathway, and the mechanisms of escape
BRAF Inhibitor Melanoma Others Tumor Types
or alternative signaling.
Vemurafenib  NCT01495988  NCT01709292: Thyroid,
locally advanced Melanoma
 NCT01813214  NCT01286753:Papillary
thyroid The success of RAF kinase inhibition was a turning point
 NCT01611675  NCT01524978: Any in the treatment of melanoma. However, as with other
BRAFV600-mutant tumor
 NCT01942993  NCT01771458: Any tumor
agents, resistance to treatment occurred and was mapped
 NCT01638676 to the MAPK/ERK pathway. Primary resistance to
 NCT01781026
 NCT01586195
vemurafenib in V600EBRAF melanomas can occur
 NCT00949702 through increased cellular proliferation in response to loss
 NCT01378975 of function of tumor suppressors or dysregulation of
 NCT01248936
Dabrafenib  NCT01682213  NCT01340846: Any mechanisms that prevent apoptosis. Phosphatase and ten-
BRAFV600-mutant tumor sin homolog (PTEN) deficiency is a major mechanism
 NCT01721603  NCT01723202: Thyroid
 NCT01266967 through which the prosurvival AKT signaling pathway
 NCT01153763  NCT01336634: NSCLC becomes constitutively activated. This was observed in
with BRAF mutation
melanoma cell lines treated with vemurafenib. This
Abbreviations: BRAF, BRAF, v-raf murine sarcoma viral oncogene homolog; PTEN deficiency was accompanied by loss of induction
NCT, National Clinical Trials identifier; V600, valine mutation at BRAF codon of the proapoptotic BIM/BCL2L11 protein and resulted
400.
in primary resistance in these cell lines.71 Selective cyto-
plasmic redistribution of the transcription factor
class, may cause abnormal skin cell proliferation, leading FOXO3a led to decreased transcription of proapoptotic
to keratoacanthomas or squamous cell cancers in approxi- proteins.85,86 These findings, coupled with the reality that
mately 10% to 20% of patients.57-59 The development of not every mutation-positive tumor will respond to B-RAF
these lesions is caused by paradoxical activation of the nor- inhibitor therapy despite activating mutations in BRAF,
mal MAPK/ERK pathway in the genomically normal skin underscores the need for future research into mechanisms
keratinocytes.59 The combination of BRAF and MEK of primary resistance.
inhibitors in metastatic melanoma resulted in improved It has been observed that tumors with oncogenic
treatment safety by counterbalancing activation of the driver mutations in the MAPK/ERK pathway progress de-
normal MAPK/ERK pathway, yielding a marked reduc- spite an initial response to targeted intervention (second-
tion in the frequency of the paradoxical oncogenic skin ary resistance). Multiple secondary mechanisms of
changes.60,61 The combination of dabrafenib (a BRAF ki- resistance have been identified in melanoma, including
nase inhibitor) with trametinib (an MEK inhibitor) new activating mutations in MAPK/ERK pathway genes87
caused keratoacanthomas in 7% of patients along with and NRAS,73 increased dimers of splice variants of wild-
rare squamous cell skin cancers compared with a fre- type BRAF,50,74,88 amplification of wild-type BRAF and
quency of 19% for dabrafenib alone.60 This safer combi- MEK,72 and increased CRAF.89 New studies have demon-
nation is now under evaluation in numerous other strated that C-RAF activates the MAPK/ERK pathway
cancers. through the acquisition of secondary mutations that
increase its half-life, avoiding degradation and allowing
MAPK/ERK Pathway Susceptibility Varies by heterodimerization with B-RAF.90 Many of these mecha-
Tumor Type nisms result in paradoxical hyperactivation of ERK.13
Different morphomolecular human tumors have demon- Intratumoral heterogeneity allows multiple mechanisms
strated unexpected differential responses to signal inter- to be identified within a single patient’s tumor, such as
ruption and may develop unique mechanisms of primary unique mutations in NRAS and an alternative splice vari-
and secondary resistance (Table 4).82-84 CRC and low- ant of BRAF.91 These findings support the concept that
grade serous ovarian cancers harbor the same mutations tumors demonstrate clonal evolution and plasticity over
that are observed in melanoma, KRAS and BRAF, but time, adapting to microenvironment and pharmacologic
have very different responses to inhibition of the exposures.92

Cancer Month 00, 2014 5

Review Article

TABLE 3. Phase 2 Trials of Mitogen-Activated predisposed to susceptibility to inhibitor therapy like

Protein Kinase Inhibitors Across Tumor Types patients with melanoma. It is now routine clinical practice
Clinical Trial to test for KRAS mutation before the initiation of EGFR
inhibitors. Thus, rather than functioning as therapeutic
MAPK Inhibitor Melanoma Other Tumor Types
targets in CRC, these genomic events in the MAPK/ERK
Selumetinib  NCT01143402  NCT00888134: Any tumor pathway are validated negative predictive biomarkers for
type EGFR inhibitor intervention.
 NCT00866177  NCT00553332: Biliary
 NCT01160718: Breast CRC resistance to B-RAF inhibition has been attrib-
 NCT00780676: Breast uted to differential activation of EGFR in the cell mem-
 NCT00514761: Colorectal
 NCT01011933: Endometrial
brane, reinforcing the differential relevance of EGFR
 NCT01089101: Glioma expression across tumor types. Treatment of BRAF-mu-
 NCT01752569: Kaposi
sarcoma
tant CRC cell lines with vemurafenib resulted in a strong
 NCT00604721: Liver increase in 1068Y-EGFR phosphorylation and receptor
 NCT00372788: NSCLC activation through inhibition of cell division cycle 25C
 NCT01306045: NSCLC,
thymic (CDC25C) phosphatase, which regulates 1068Y-EGFR
 NCT00372944: Pancreatic phosphorylation. Blockade of MAPK/ERK by BRAF or
 NCT00551070: Ovarian,
peritoneum MEK inhibitors prevented CDC25C activation, resulting
 NCT00559949: Papillary in increased 1068Y-EGFR and subsequent activation of
thyroid
 NCT01843062: Thyroid
other downstream pathways, such AKT. EGFR suppres-
Trametinib  NCT01978236  NCT01827384: Any tumor sion combined with vemurafenib markedly inhibited pro-
type
 NCT01037127  NCT01943864: Biliary
liferation in CRC cells and may be a mechanism to
 NCT01328106  NCT01553851: Oral increase clinical activity.76
squamous Cross-communication between the MAPK/ERK
BRAF inhibitor  LCCC 1128:  NCT01723202: Thyroid
plus trametinib NCT01726738 pathway and parallel pathways, such as the PI3K/AKT
 NCT01072175  NCT01750918: Colorectal and wingless-type/calcium (Wnt-Ca21) pathways, is criti-
 NCT01972347
 NCT01928940 cal to abnormal proliferation and therapy resistance.
 NCT01619774 These parallel pathways are activated when the MAPK/
 NCT01978236
 NCT01915602:
ERK pathway is attenuated, and they drive cellular prolif-
Hepatocellular eration. The inhibition of PI3K or AKT or the use of
Pimasertib  NCT01693068  NCT01936363L Ovarian
Refametinib —  NCT01915589:
hypomethylating agents that secondarily block AKT sig-
Hepatocellular naling can overcome this mechanism of resistance in
 NCT01915602: vitro.78 Understanding of mechanisms of induction of
Hepatocellular
parallel signaling is needed to guide development of com-
Abbreviations: BRAF, v-raf murine sarcoma viral oncogene homolog; bination therapies. Recently, Spreafico et al demonstrated
MAPK, mitogen-activated protein kinase; NCT, National Clinical Trials iden-
tifier; NSCLC, nonsmall cell lung cancer.
a potential role of the noncanonical Wnt/Ca21 signaling
pathway in overcoming resistance of CRC to MEK inhib-
itors using cyclosporine (a Wnt/Ca11 modulator) in a
CRC model of patient-derived tumor xenografts.79 Those mod-
Targeted kinase inhibitors (TKIs) that have been benefi- els demonstrated that drug combinations blocking both a
cial in melanoma have not yielded similar activity in targeted pathway and its associated counter-regulatory
patients with CRC. RAF signaling is downstream of RAS signal can effectively abrogate resistance of CRC to BRAF
in the MAPK/ERK pathway, such that the presence of or MEK inhibitors.
BRAF and KRAS mutations in CRC should lead to sensi-
tivity to RAF-targeted agents and circumvent the inhibi- Ovarian cancer
tion of upstream signals, such as those emanating from Ovarian cancers can be classified into distinct types, some
receptor kinases. Consistent with the latter expectation, of which are characterized by genetic mutations that may
investigators demonstrated that BRAF and KRAS muta- involve the MAPK/ERK pathway. Type II ovarian cancers
tions were negative predictors of deriving a benefit from include high-grade serous tumors, and defects in DNA
cetuximab and panitumumab (EGFR inhibitor therapy) repair through the loss of normal p53 regulation are
in phase 3 clinical trials.93,94 Those patients were not observed in almost all tumors.95,96 Type I ovarian tumors

6 Cancer Month 00, 2014

 Tissue-Specific MAPK Signaling/Burotto et al

TABLE 4. Mechanisms of Primary and Secondary Resistance to Tyrosine Kinase Inhibitors in Different Tumor
Types

Mechanism of Resistance (References)

Tumor Primary Secondary

Lung MET amplification (Turke 201062), BIM polymor- T790M mutation (Kobayashi 200565), EGFR ampli-
phism (Ng 201263 and Nakagawa 201364) fication (Sequist 201166), Her2 amplification
(Takezawa 201267), PIK3CA mutations (Sequist
201166), MET amplification (Engelman 200768)
Melanoma NF1 loss (Whittaker 201369 and Maertens 201370), BRAF amplification (Corcoran 201072), NRAS
PTEN loss (Paraiso 201171) amplification, increase in CRAF (Montagut
200873), splice variant BRAF (Poulikakos
201174), increased activation of AKT (Atefi
201175), NF1 loss (Whittaker 201369)
Colorectal EGFR activation (Prahallad 201276 and Corcoran NA
201277), PI3K/AKT activation (Mao 201378), Wnt/
Ca21 activation (Spreafico 201379)
Ovarian PI3K activation (Sheppard 201380), activation of NA
ERa (Hou 201381)

Abbreviations: BIM, B-cell chronic lymphocytic leukemia/lymphoma (Bcl-2)-like 11; BRAF, v-Raf murine sarcoma viral oncogene homolog B1; CRAF, RAF
proto-oncogene serine/threonine-protein kinase; EGFR, epidermal growth factor receptor; ERa, estrogen receptor a; Her2, human epidermal growth factor
receptor 2; MET, mesenchymal epithelial transition factor; NA, not applicable; NF1, nuclear factor 1; NRAS, neuroblastoma RAS viral oncogene homolog;
PI3K, phosphatidylinositol-3-kinase; PTEN, phosphatase and tensin homolog; T790M, threonine to methionine substitution at position 790; Wnt, wingless
type.

Figure 2. Mechanisms of resistance to v-raf murine sarcoma viral oncogene homolog B1 (B-Raf) inhibitors are illustrated. (a) In
melanoma, mechanisms of secondary resistance to BRAF inhibitors include the expression of BRAF splice variants, RAF proto-
oncogene serine/threonine-protein kinase (C-Raf) activation (all of which activates mitogen-activated protein kinase/extracellular
signal-regulating kinase [MAPK/ERK]), or signaling through alternative pathways like protein kinase B/mammalian target of rapa-
mycin (AKT/mTOR), etc (see Table 4). N-Ras indicates neuroblastoma RAS viral oncogene homolog; V600E, a valine to glutamic
acid substitution at BRAF position 600; PI3 kinase, phosphatidylinositol-3-kinase; MEK, mitogen-activated protein kinase-kinase.
(b) In colorectal cancer, primary resistance to BRAF inhibitors is caused by direct activation of epidermal growth factor receptor
(EGFR) and the AKT/mTOR pathway. CDC25C, indicates cell division cycle 25C; WNT, wingless type.

include low-grade serous and endometrioid, clear cell,
mucinous, and borderline tumors (BOTs); low-grade se-
rous tumors have mutations in KRAS (27%-36%), BRAF
(33%-50%), and PIK3CA; whereas nearly all mucinous
tumors may have KRAS mutations.41,42,97 One study
reported that 57% of BOTs or low-grade serous tumors
had V600BRAF or KRAS codon 12 mutations. It is inter-
esting to note that, in 1 study, patients who had BRAF
mutations had no recurrence after a median follow-up of
3.6 years.98 Ho et al observed KRAS or BRAF mutations
in 86% of cystadenomas adjacent to BOTs, and 88% of
BOTs had mutations.99 There is a loss of frequency of

Cancer Month 00, 2014 7

Review Article
BRAF and KRAS mutations in the transition from nonma-
lignant to malignant disease, from cystadenoma or BOT,
to invasive low-grade serous cancer. The mechanism of
this selective process, in which there is loss of an otherwise
recognized oncogenic mutation during the process of ac-
quisition of an invasive phenotype, is unknown. This is
the only example we identified in which there was such
loss of a perceived gain-of-function mutation.
The identification of these mutations led to the logi-
cal hypothesis that such ovarian neoplasms were a new
frontier for experimentation with targeted BRAF and
MEK inhibitor therapy. Gynecologic Oncology Group
Study 239, which was a phase 2 trial of the MEK inhibitor
selumetinib in 52 previously treated patients with
low-grade serous ovarian tumors, yielded a 15% overall
response rate and a median progression-free survival of 11
months. This was compared with an historical
progression-free survival of 7 months. Mutational analy-
ses were performed in tumors from 34 patients, and KRAS
and BRAF mutations were found in 41% and 6%, respec-
tively, although mutations did not correlate with response
or longer progression-free survival.100 These examples
argue against a preponderant role of the MAPK/ERK
pathway as a targeting oncogenic driver in these tumors
despite the presence of mutations. Studies of sorafenib
predominantly in patients with recurrent, high-grade
serous ovarian cancer did not demonstrate biochemical
activity of reduced ERK activation pretreatment or on-
treatment, perhaps consistent with the lack of genomic
events in the MAPK/ERK pathway in those tumors.101
Interactions between the MAPK/ERK pathway and
estrogen receptor-a (ERa) have also been identified in
preclinical studies. MEK inhibition caused an increase in
ERa expression independent of AKT signaling in ovar-
ian cell lines that were positive for ERa. The addition of
the ER inhibitor fulvestrant caused synergistic suppres-
sion of tumor growth in vitro and in an in vivo model.81
This may be a direction for clinical study using modula-
tion of the MAPK/ERK pathway to secondarily regulate
a parallel pathway. This reinforces how ovarian cancer is
a challenging environment in which to study the tumor-
specific effects of MAPK/ERK pathway activation. Its
broad range of cellular diversity and complexity of path-
way activation lends itself to combination therapy, neces-
sitating greater understanding of the interaction of the
pathways.
Conclusion
The downstream MAPK/ERK signaling node, which is
activated predominantly by upstream SRC/RAS/RAF
8 Cancer
signaling, is also regulated by modulation through parallel
pathways. This creates a complexity within and between
tumors that impedes the ability to translate therapeutic
findings across tumors. Tissue and subtype specificity in
signaling adds a level of complexity to the application of
novel targeted agents, even against an otherwise dominant
pathway. The MAPK/ERK pathway stimulates cellular
proliferation and invasion; however, its activation also can
increase cellular apoptosis or antagonize pro-oncogenic
input from other signals. The MAPK/ERK pathway dem-
onstrates both oncogene and tumor suppressor, effects
depending on the tissue-specific tumor microenviron-
ment. Although cancers share common mutations, differ-
ent cell types have developed unique responses to the
mutations. These mutations may behave as oncogenic
drivers, passenger mutations, or regulatory events. The
role of the MAPK/ERK pathway in the tumor microen-
vironment has long been recognized. This pathway is crit-
ical in the process of physiologic and malignant invasion
and angiogenesis; and, most recently, a clear role for
MAPK/ERK has been demonstrated in tumor-immune
system interactions. Hence, MAPK/ERK activation is a
multifaceted target under varied regulatory bodies. Regu-
latory mechanisms may lead to the activation of alterna-
tive pathways and paradoxical hyperactivation of the
normal MAPK/ERK pathway. One unintended and
unexpected consequence of KRAS/BRAF inhibitor drug
therapy is increased activity of the normal MAPK/ERK
pathway, which can lead to the development of secondary
malignancies. Some novel combination therapies have
demonstrated increased treatment efficacy by addressing
both a specific target and its counter-regulatory effect in
the complex milieu of cellular signaling. In shaping future
approaches toward personalized medicine, the challenge is
clear: we must strike a delicate balance between exploiting
shared genetic targets and acknowledging the unique fea-
tures of human cancers.
FUNDING SUPPORT
This work was supported by the Intramural Program of the Center
for Cancer Research, National Cancer Institute, National Institutes
of Health, USA.
CONFLICT OF INTEREST DISCLOSURES
The authors made no disclosures.
REFERENCES
1. Hudson TJ, Anderson W, Artez A, et al. International network of
cancer genome projects. Nature. 2010;464:993-998.
2. Ciriello G, Miller ML, Aksoy BA, Senbabaoglu Y, Schultz N,
Sander C. Emerging landscape of oncogenic signatures across
human cancers. Nat Genet. 2013;45:1127-1133.
Month 00, 2014

3. Weinstein IB, Joe A. Oncogene addiction. Cancer Res. 2008;68:
3077-3080; discussion 3080.
4. Hanahan D, Weinberg RA. Hallmarks of cancer: the next genera-
tion. Cell. 2011;144:646-674.
5. De Luca A, Maiello MR, D’Alessio A, Pergameno M, Normanno
N. The RAS/RAF/MEK/ERK and the PI3K/AKT signalling path-
ways: role in cancer pathogenesis and implications for therapeutic
approaches. Expert Opin Ther Targets. 2012;16(suppl 2):S17-S27.
6. Yang SH, Sharrocks AD, Whitmarsh AJ. MAP kinase signalling
cascades and transcriptional regulation. Gene. 2013;513:1-13.
7. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr,
Kinzler KW. Cancer genome landscapes. Science. 2013;339:1546-
1558.
8. Tebbutt N, Pedersen MW, Johns TG. Targeting the ERBB family
in cancer: couples therapy. Nat Rev Cancer. 2013;13:663-673.
9. Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat
Rev Cancer. 2003;3:459-465.
10. Muram-Zborovski TM, Stevenson DA, Viskochil DH, Dries DC,
Wilson AR, Rong M. SPRED 1 mutations in a neurofibromatosis
clinic. J Child Neurol. 2010;25:1203-1209.
11. Wan PT, Garnett MJ, Roe SM, et al. Mechanism of activation of
the RAF-ERK signaling pathway by oncogenic mutations of
B-RAF. Cell. 2004;116:855-867.
12. Farooq A, Zhou MM. Structure and regulation of MAPK phospha-
tases. Cell Signal. 2004;16:769-779.
13. Cossa G, Gatti L, Cassinelli G, Lanzi C, Zaffaroni N, Perego P.
Modulation of sensitivity to antitumor agents by targeting the
MAPK survival pathway. Curr Pharm Des. 2013;19:883-894.
14. Dhanasekaran N, Premkumar Reddy E. Signaling by dual specific-
ity kinases. Oncogene. 1998;17:1447-1455.
15. Robinson MJ, Cobb MH. Mitogen-activated protein kinase path-
ways. Curr Opin Cell Biol. 1997;9:180-186.
16. Pullikuth AK, Catling AD. Scaffold mediated regulation of MAPK
signaling and cytoskeletal dynamics: a perspective. Cell Signal.
2007;19:1621-1632.
17. Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP.
Phosphorylation and functional inactivation of TSC2 by Erk implica-
tions for tuberous sclerosis and cancer pathogenesis. Cell. 2005;121:
179-193.
18. Chu B, Soncin F, Price BD, Stevenson MA, Calderwood SK.
Sequential phosphorylation by mitogen-activated protein kinase and
glycogen synthase kinase 3 represses transcriptional activation by
heat shock factor-1. J Biol Chem. 1996;271:30847-30857.
19. Schmid RS, Pruitt WM, Maness PF. A MAP kinase-signaling path-
way mediates neurite outgrowth on L1 and requires Src-dependent
endocytosis. J Neurosci. 2000;20:4177-4188.
20. Wolf I, Rubinfeld H, Yoon S, Marmor G, Hanoch T, Seger R.
Involvement of the activation loop of ERK in the detachment from
cytosolic anchoring. J Biol Chem. 2001;276:24490-24497.
21. Zassadowski F, Rochette-Egly C, Chomienne C, Cassinat B. Regu-
lation of the transcriptional activity of nuclear receptors by the
MEK/ERK1/2 pathway. Cell Signal. 2012;24:2369-2377.
22. Sigoillot FD, Evans DR, Guy HI. Growth-dependent regulation of
mammalian pyrimidine biosynthesis by the protein kinase A and
MAPK signaling cascades. J Biol Chem. 2002;277:15745-15751.
23. Arthur JS, Ley SC. Mitogen-activated protein kinases in innate im-
munity. Nat Rev Immunol. 2013;13:679-692.
24. Winters ME, Mehta AI, Petricoin EF 3rd, Kohn EC, Liotta LA.
Supra-additive growth inhibition by a celecoxib analogue and
carboxyamido-triazole is primarily mediated through apoptosis.
Cancer Res. 2005;65:3853-3860.
25. Murphy LO, Smith S, Chen RH, Fingar DC, Blenis J. Molecular
interpretation of ERK signal duration by immediate early gene
products. Nat Cell Biol. 2002;4:556-564.
26. Murphy T, Hori S, Sewell J, Gnanapragasam VJ. Expression and
functional role of negative signalling regulators in tumour develop-
ment and progression. Int J Cancer. 2010;127:2491-2499.
27. Katz M, Amit I, Yarden Y. Regulation of MAPKs by growth factors and
receptor tyrosine kinases. Biochim Biophys Acta. 2007;1773:1161-1176.
Cancer Month 00, 2014
Tissue-Specific MAPK Signaling/Burotto et al
28. Deschenes-Simard X, Gaumont-Leclerc MF, Bourdeau V, et al.
Tumor suppressor activity of the ERK/MAPK pathway by promot-
ing selective protein degradation. Genes Dev. 2013;27:900-915.
29. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Onco-
genic ras provokes premature cell senescence associated with accu-
mulation of p53 and p16INK4a. Cell. 1997;88:593-602.
30. Bric A, Miething C, Bialucha CU, et al. Functional identification
of tumor-suppressor genes through an in vivo RNA interference
screen in a mouse lymphoma model. Cancer Cell. 2009;16:324-
335.
31. Colombino M, Capone M, Lissia A, et al. BRAF/NRAS mutation
frequencies among primary tumors and metastases in patients with
melanoma. J Clin Oncol. 2012;30:2522-2529.
32. Edlundh-Rose E, Egyhazi S, Omholt K, et al. NRAS and BRAF
mutations in melanoma tumours in relation to clinical characteris-
tics: a study based on mutation screening by pyrosequencing. Mela-
noma Res. 2006;16:471-478.
33. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene
in human cancer. Nature. 2002;417:949-954.
34. Murugan AK, Dong J, Xie J, Xing M. MEK1 mutations, but not
ERK2 mutations, occur in melanomas and colon carcinomas, but
none in thyroid carcinomas. Cell Cycle. 2009;8:2122-2124.
35. Nikolaev SI, Rimoldi D, Iseli C, et al. Exome sequencing identifies
recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma.
Nat Genet. 2012;44:133-139.
36. Seo JS, Ju YS, Lee WC, et al. The transcriptional landscape and muta-
tional profile of lung adenocarcinoma. Genome Res. 2012;22:2109-
2119.
37. Cardarella S, Ogino A, Nishino M, et al. Clinical, pathologic, and
biologic features associated with BRAF mutations in non-small cell
lung cancer. Clin Cancer Res. 2013;19:4532-4540.
38. Cancer Genome Atlas Network. Comprehensive molecular character-
ization of human colon and rectal cancer. Nature. 2012;487:330-
337.
39. Tol J, Nagtegaal ID, Punt CJ. BRAF mutation in metastatic colo-
rectal cancer. N Engl J Med. 2009;361:98-99.
40. Sieben NL, Macropoulos P, Roemen GM, et al. In ovarian neo-
plasms, BRAF, but not KRAS, mutations are restricted to low-
grade serous tumours. J Pathol. 2004;202:336-340.
41. Bell DA. Origins and molecular pathology of ovarian cancer. Mod
Pathol. 2005;18(suppl 2):S19-S32.
42. Singer G, Oldt R 3rd, Cohen Y, et al. Mutations in BRAF and
KRAS characterize the development of low-grade ovarian serous
carcinoma. J Natl Cancer Inst. 2003;95:484-486.
43. Bansal M, Gandhi M, Ferris RL, et al. Molecular and histopatho-
logic characteristics of multifocal papillary thyroid carcinoma. Am J
Surg Pathol. 2013;37:1586-1591.
44. Xing M, Alzahrani AS, Carson KA, et al. Association between
BRAF V600E mutation and mortality in patients with papillary
thyroid cancer. JAMA. 2013;309:1493-1501.
45. Cohen Y, Xing M, Mambo E, et al. BRAF mutation in papillary
thyroid carcinoma. J Natl Cancer Inst. 2003;95:625-647.
46. Xi L, Arons E, Navarro W, et al. Both variant and IGHV4-34-
expressing hairy cell leukemia lack the BRAF V600E mutation.
Blood. 2012;119:3330-3332.
47. Tiacci E, Trifonov V, Schiavoni G, et al. BRAF mutations in
hairy-cell leukemia. N Engl J Med. 2011;364:2305-2315.
48. Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-acti-
vated protein kinase cascade for the treatment of cancer. Oncogene.
2007;26:3291-3310.
49. Bedard PL, Hansen AR, Ratain MJ, Siu LL. Tumour heterogeneity
in the clinic. Nature. 2013;501:355-364.
50. Poulikakos PI, Zhang C, Bollag G, Shokat KM, Rosen N. RAF
inhibitors transactivate RAF dimers and ERK signalling in cells
with wild-type BRAF. Nature. 2010;464:427-430.
51. Solit DB, Garraway LA, Pratilas CA, et al. BRAF mutation predicts
sensitivity to MEK inhibition. Nature. 2006;439:358-362.
52. Bollag G, Hirth P, Tsai J, et al. Clinical efficacy of a RAF inhibitor
needs broad target blockade in BRAF-mutant melanoma. Nature.
2010;467:596-599.
9
Review Article
53. Boni A, Cogdill AP, Dang P, et al. Selective BRAFV600E inhibi-
tion enhances T-cell recognition of melanoma without affecting
lymphocyte function. Cancer Res. 2010;70:5213-5219.
54. Bekaii-Saab T, Phelps MA, Li X, et al. Multi-institutional phase II
study of selumetinib in patients with metastatic biliary cancers. J
Clin Oncol. 2011;29:2357-2363.
55. Ho AL, Grewal RK, Leboeuf R, et al. Selumetinib-enhanced radioio-
dine uptake in advanced thyroid cancer. N Engl J Med. 2013;368:623-
632.
56. Janne PA, Shaw AT, Pereira JR, et al. Selumetinib plus docetaxel
for KRAS-mutant advanced non-small-cell lung cancer: a rando-
mised, multicentre, placebo-controlled, phase 2 study. Lancet Oncol.
2013;14:38-47.
57. Sosman JA, Kim KB, Schuchter L, et al. Survival in BRAF V600-
mutant advanced melanoma treated with vemurafenib. N Engl J
Med. 2012;366:707-714.
58. Hauschild A, Grob JJ, Demidov LV, et al. Dabrafenib in BRAF-
mutated metastatic melanoma: a multicentre, open-label, phase 3
randomised controlled trial. Lancet. 2012;380:358-365.
59. Vin H, Ching G, Ojeda SS, et al. Sorafenib suppresses JNK-
dependent apoptosis through inhibition of ZAK. Mol Cancer Ther.
2014;13:221-229.
60. Flaherty KT, Infante JR, Daud A, et al. Combined BRAF and
MEK inhibition in melanoma with BRAF V600 mutations. N Engl
J Med. 2012;367:1694-1703.
61. US Food and Drug Administration. Trametinib and Dabrafenib
approval. Washington, DC: US Food and Drug Administration;
2014.
62. Turke AB, Zejnullahu K, Wu YL, et al. Preexistence and clonal
selection of MET amplification in EGFR mutant NSCLC. Cancer
Cell. 2010;17:77-88.
63. Ng KP, Hillmer AM, Chuah CT, et al. A common BIM deletion
polymorphism mediates intrinsic resistance and inferior responses
to tyrosine kinase inhibitors in cancer. Nat Med. 2012;18:521-528.
64. Nakagawa T, Takeuchi S, Yamada T, et al. EGFR-TKI resistance
due to BIM polymorphism can be circumvented in combination
with HDAC inhibition. Cancer Res. 2013;73:2428-2434.
65. Kobayashi S, Boggon TJ, Dayaram T, et al. EGFR mutation and
resistance of non-small-cell lung cancer to gefitinib. N Engl J Med.
2005;352:786-792.
66. Sequist LV, Waltman BA, Dias-Santagata D, et al. Genotypic and
histological evolution of lung cancers acquiring resistance to EGFR
inhibitors [serial online]. Sci Transl Med. 2011;3:75ra26.
67. Takezawa K, Pirazzoli V, Arcila ME, et al. HER2 amplification: a
potential mechanism of acquired resistance to EGFR inhibition in
EGFR-mutant lung cancers that lack the second-site EGFRT790M
mutation. Cancer Discov. 2012;2:922-933.
68. Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET amplifica-
tion leads to gefitinib resistance in lung cancer by activating
ERBB3 signaling. Science. 2007;316:1039-1043.
69. Whittaker SR, Theurillat JP, Van Allen E, et al. A genome-scale
RNA interference screen implicates NF1 loss in resistance to RAF
inhibition. Cancer Discov. 2013;3:350-362.
70. Maertens O, Johnson B, Hollstein P, et al. Elucidating distinct
roles for NF1 in melanoma genesis. Cancer Discov. 2013;3:338-
349.
71. Paraiso KH, Xiang Y, Rebecca VW, et al. PTEN loss confers
BRAF inhibitor resistance to melanoma cells through the suppres-
sion of BIM expression. Cancer Res. 2011;71:2750-2760.
72. Corcoran RB, Dias-Santagata D, Bergethon K, Iafrate AJ,
Settleman J, Engelman JA. BRAF gene amplification can promote
acquired resistance to MEK inhibitors in cancer cells harboring the
BRAF V600E mutation [serial online]. Sci Signal 2010;3:ra84.
73. Montagut C, Sharma SV, Shioda T, et al. Elevated CRAF as a
potential mechanism of acquired resistance to BRAF inhibition in
melanoma. Cancer Res. 2008;68:4853-4861.
74. Poulikakos PI, Persaud Y, Janakiraman M, et al. RAF inhibitor
resistance is mediated by dimerization of aberrantly spliced
BRAF(V600E). Nature. 2011;480:387-390.
10
75. Atefi M, von Euw E, Attar N, et al. Reversing melanoma cross-
resistance to BRAF and MEK inhibitors by co-targeting the AKT/
mTOR pathway [serial online]. PLoS One. 2011;6:e28973.
76. Prahallad A, Sun C, Huang S, et al. Unresponsiveness of colon
cancer to BRAF(V600E) inhibition through feedback activation of
EGFR. Nature. 2012;483:100-103.
77. Corcoran RB, Ebi H, Turke AB, et al. EGFR-mediated re-
activation of MAPK signaling contributes to insensitivity of BRAF
mutant colorectal cancers to RAF inhibition with vemurafenib.
Cancer Discov. 2012;2:227-235.
78. Mao M, Tian F, Mariadason JM, et al. Resistance to BRAF inhibi-
tion in BRAF-mutant colon cancer can be overcome with PI3K in-
hibition or demethylating agents. Clin Cancer Res. 2013;19:657-
667.
79. Spreafico A, Tentler JJ, Pitts TM, et al. Rational combination of a
MEK inhibitor, selumetinib, and the Wnt/calcium pathway
modulator, cyclosporin A, in preclinical models of colorectal can-
cer. Clin Cancer Res. 2013;19:4149-4162.
80. Sheppard KE, Cullinane C, Hannan KM, et al. Synergistic inhibi-
tion of ovarian cancer cell growth by combining selective PI3K/
mTOR and RAS/ERK pathway inhibitors. Eur J Cancer. 2013;49:
3936-3944.
81. Hou JY, Rodriguez-Gabin A, Samaweera L, et al. Exploiting MEK
inhibitor-mediated activation of ERalpha for therapeutic interven-
tion in ER-positive ovarian carcinoma [serial online]. PLoS One.
2013;8:e54103.
82. Thomas RK, Baker AC, Debiasi RM, et al. High-throughput
oncogene mutation profiling in human cancer. Nat Genet. 2007;39:
347-351.
83. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epi-
dermal growth factor receptor underlying responsiveness of non-
small-cell lung cancer to gefitinib. N Engl J Med. 2004;350:2129-
2139.
84. Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung
adenocarcinomas to gefitinib or erlotinib is associated with a second
mutation in the EGFR kinase domain [serial online]. PLoS Med.
2005;2:e73.
85. Yang JY, Hung MC. A new fork for clinical application: targeting
forkhead transcription factors in cancer. Clin Cancer Res. 2009;15:
752-757.
86. Fedorenko IV, Paraiso KH, Smalley KS. Acquired and intrinsic
BRAF inhibitor resistance in BRAF V600E mutant melanoma. Bio-
chem Pharmacol. 2011;82:201-209.
87. Wang H, Daouti S, Li WH, et al. Identification of the
MEK1(F129L) activating mutation as a potential mechanism of
acquired resistance to MEK inhibition in human cancers carrying
the B-RafV600E mutation. Cancer Res. 2011;71:5535-5545.
88. Roskoski R Jr. RAF protein-serine/threonine kinases: structure and
regulation. Biochem Biophys Res Commun. 2010;399:313-317.
89. Rosell R, Bivona TG, Karachaliou N. Genetics and biomarkers in
personalisation of lung cancer treatment. Lancet. 2013;382:720-
731.
90. Antony R, Emery CM, Sawyer AM, Garraway LA. C-RAF
mutations confer resistance to RAF inhibitors. Cancer Res. 2013;73:
4840-4851.
91. Romano E, Pradervand S, Paillusson A, et al. Identification of mul-
tiple mechanisms of resistance to vemurafenib in a patient with
BRAFV600E-mutated cutaneous melanoma successfully rechal-
lenged after progression. Clin Cancer Res. 2013;19:5749-5757.
92. Meacham CE, Morrison SJ. Tumour heterogeneity and cancer cell
plasticity. Nature. 2013;501:328-337.
93. Di Nicolantonio F, Martini M, Molinari F, et al. Wild-type BRAF
is required for response to panitumumab or cetuximab in meta-
static colorectal cancer. J Clin Oncol. 2008;26:5705-5712.
94. Richman SD, Seymour MT, Chambers P, et al. KRAS and BRAF
mutations in advanced colorectal cancer are associated with poor
prognosis but do not preclude benefit from oxaliplatin or irinotecan:
results from the MRC FOCUS trial. J Clin Oncol. 2009;27:5931-
5937.
95. Kohn EC, Romano S, Lee JM. Clinical implications of using molecular
diagnostics for ovarian cancers. Ann Oncol. 2013;24(suppl 10):x22-x26.
Cancer Month 00, 2014

96. Lee JM, Ledermann JA, Kohn EC. PARP Inhibitors for BRCA1/2
mutation-associated and BRCA-like malignancies. Ann Oncol.
2014;25:32-40.
97. Shih Ie M, Kurman RJ. Ovarian tumorigenesis: a proposed model
based on morphological and molecular genetic analysis. Am J
Pathol. 2004;164:1511-1518.
98. Grisham RN, Iyer G, Garg K, et al. BRAF mutation is associated
with early stage disease and improved outcome in patients with
low-grade serous ovarian cancer. Cancer. 2013;119:548-554.
Cancer Month 00, 2014
Tissue-Specific MAPK Signaling/Burotto et al
99. Ho CL, Kurman RJ, Dehari R, Wang TL, Shih Ie M. Mutations
of BRAF and KRAS precede the development of ovarian serous
borderline tumors. Cancer Res. 2004;64:6915-6918.
100. Farley J, Brady WE, Vathipadiekal V, et al. Selumetinib in women
with recurrent low-grade serous carcinoma of the ovary or perito-
neum: an open-label, single-arm, phase 2 study. Lancet Oncol.
2013;14:134-140.
101. Matei D, Sill MW, Lankes HA, et al. Activity of sorafenib in
recurrent ovarian cancer and primary peritoneal carcinomatosis: a
gynecologic oncology group trial. J Clin Oncol. 2011;29:69-75.
11