ABSTRACT Colon cancer has been proposed to be sustained by a small subpopulation of stem-like cells with unique properties allowing them to survive conventional therapies and drive tumor

recurrence. Identification of targetable signaling pathways contributing to malignant stem-like cell maintenance may therefore translate into new therapeutic strategies to overcome drug

resistance. Here we demonstrated that MEK5/ERK5 signaling activation is associated with stem-like malignant phenotypes. Conversely, using a panel of cell line-derived three-dimensional

models, we showed that ERK5 inhibition markedly suppresses the molecular and functional features of colon cancer stem-like cells. Particularly, pharmacological inhibition of ERK5 using

expression and NF-κB transcriptional activity, suggesting a possible ERK5/NF-κB/IL-8 signaling axis regulating stem-like cell malignancy. Taken together, our results provide proof of

principle that ERK5-targeted inhibition may be a promising therapeutic approach to eliminate drug-resistant cancer stem-like cells and improve colon cancer treatment. SIMILAR CONTENT BEING

VIEWED BY OTHERS CANCER STEM CELLS: ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR CANCER THERAPY Article Open access 05 July 2024 CONTINUOUS FORMATION OF SMALL CLUSTERS WITH LGR5-POSITIVE CELLS

CONTRIBUTES TO TUMOR GROWTH IN A COLORECTAL CANCER XENOGRAFT MODEL Article 29 July 2020 IGF-1-MEDIATED FOXC1 OVEREXPRESSION INDUCES STEM-LIKE PROPERTIES THROUGH UPREGULATING CBX7 AND IGF-1R

IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA Article Open access 27 February 2024 INTRODUCTION The identification of stem-like cells within tumors has reshaped our understanding of cancer

development, introducing an additional layer of complexity to the concept of intratumoral heterogeneity1. The existence of cancer stem cells (CSCs) was demonstrated in several solid tumors,

including colon cancer2,3,4. Importantly, CSC populations are characterized by their remarkable potential to perpetuate themselves through self-renewal, while retaining the ability to

differentiate into the full repertoire of neoplastic cells forming the heterogeneous tumor mass5. Owing to their highly tumorigenic and adaptable phenotype, colon CSCs are currently

recognized as the only subset of neoplastic cells holding attributes for tumor initiation, sustained growth, and metastasis formation6. Moreover, colon CSCs show increased resistance to

conventional antitumor regimens7,8,9,10,11, arising as particularly well-suited feeders of tumor regrowth and relapse after initial response to chemotherapy6. Adding to the clinical

implications of the CSC concept, expression of stemness-associated signatures is associated with worse clinical outcomes in colon cancer patients12,13,14. Elucidation of the molecular

players regulating stem-like cell maintenance in colon cancer may therefore translate into new therapeutic strategies to overcome drug resistance and avoid tumor recurrence. Malignant

stem-like cells reproduce many of the signaling programs employed during embryonic development and tissue homeostasis15. The extracellular signal-regulated kinase 5 (ERK5 or BMK1) is a

non-redundant member of the mitogen-activated protein kinase (MAPK) family that operates within an exclusive MAPK kinase 5 (MEK5)-ERK5 axis to control cell proliferation, survival,

differentiation, and motility16. Targeted deletion of _Mek5_ and _Erk5_ in mice provided the first evidence for their essential role in development, leading to embryonic lethality at

mid-gestation due to defective endothelial cell function and cardiovascular formation17,18,19,20. In addition, MEK5/ERK5 signaling has been implicated in the regulation of

neurogenic21,22,23,24, myogenic25,26, and hematopoietic27,28,29 differentiation and lineage commitment. Mechanistically, ERK5 was proposed to act independently to maintain naive pluripotency

and control cell fate decisions in mouse embryonic stem cells, suggesting multiple critical functions for this kinase during differentiation30. In the intestine, activation of ERK5 is

triggered as a bypass route to rescue epithelial cell turnover upon _Erk1/2_ ablation31; however, the physiological relevance of this cascade in the gastrointestinal tract remains to be

elucidated32. On the other hand, substantial attention has been given to the link between aberrant MEK5/ERK5 signaling and the pathogenesis of colon cancer33,34,35,36. Dysregulation of both

MEK5 and ERK5 in human tumor samples is associated with more aggressive and metastatic stages of the disease33,34,35, and poorer survival rates34,35,36. Moreover, evidence from different

experimental models showed that ERK5-mediated signaling promotes tumor development, metastasis, and chemoresistance37, recapitulating the aforementioned features of colon CSCs6. However,

thus far, no relationship has been established between colon cancer stem-like phenotypes and MEK5/ERK5 signaling. In the present study, we show that MEK5/ERK5 signaling contributes to

sustained stemness in colon cancer, at least in part, through the activation of a downstream NF-κB/IL-8 axis. More importantly, we provide evidence that pharmacological inhibition of ERK5

may be a promising therapeutic approach to eliminate malignant stem-like cells, avoid chemotherapy resistance, and improve colon cancer treatment. RESULTS MEK5/ERK5 SIGNALING ACTIVATION

CORRELATES WITH COLON CANCER STEM-LIKE CELL PHENOTYPES Three-dimensional sphere models are widely used to selectively promote the growth of tumor cell populations with stem-like

properties38,39, representing a functional system for the in vitro discovery of new signaling pathways regulating self-renewal and differentiation in CSCs. In the present study, we used a

panel of established human colon cancer cell lines to generate sphere cultures. For this purpose, cells were grown in non-adherent conditions, using serum-free medium supplemented with

growth factors. Under this experimental setting, only malignant cells with stem cell features are expected to survive and proliferate, giving rise to free-floating multicellular spheres,

also known as tumorspheres38,39. After 1 week, HCT116, HT29, SW480, and SW620 cells were shown to efficiently form tumorspheres (Supplementary Figure S1a), which is in agreement with

previous observations40,41,42. Additionally, the expression levels of genes involved in intestinal cell differentiation, including _BMP4_, _CDX2_, _AQP3_, and _ADA_, were significantly

decreased in tumorsphere cultures, as compared with their adherent counterparts (_p_ < 0.05) (Supplementary Figure S1b). On the other hand, the expression profile of the

stemness-associated transcripts _SOX2_, _NANOG_, _OCT4_, and _BMI1_ was mostly enriched (_p_ < 0.05), further confirming that sphere-forming populations were enriched for undifferentiated

cells. To determine whether MEK5/ERK5 signaling may be a relevant player in colon cancer stem-like cells, we first analyzed the activation status of these kinases in tumorsphere and matched

adherent cultures. Immunoblot analysis showed that, except for HCT116-derived tumorspheres, colon cancer cells grown as spheres had significantly higher levels of MEK5 phosphorylation,

compared with monolayer-cultured cells (_p_ < 0.05) (Fig. 1a, upper panel). Further, ERK5 phosphorylation was shown to be consistently increased in tumorsphere cultures across all cell

lines tested (_p_ < 0.01) (Fig. 1a, lower panel), validating that MEK5/ERK5 signaling is overactivated in neoplastic populations enriched for stem-like cells. In turn, forced activation

of ERK5 by ectopic expression of a constitutively active mutant of MEK5 (CA-MEK5) in SW480 adherent cultures (Fig. 1b) was associated with lower expression of genes involved in

differentiation, and higher levels of stem cell markers, relative to empty vector control cells (_p_ < 0.05) (Fig. 1c). Changes in NANOG, OCT4, and SOX2 were confirmed at the protein

level (Fig. 1d). Together, these findings demonstrate that MEK5/ERK5 activation correlates with a shift toward an undifferentiated state in colon cancer cells, suggesting that colon cancer

stem-like populations may be dependent on ERK5-mediated signaling. ERK5 INHIBITION SUPPRESSES COLON CANCER STEM-LIKE CELL PROPERTIES To address the functional role of MEK5/ERK5 signaling in

colon cancer stem-like cells, HCT116, HT29, SW480, and SW620 cells were plated as tumorspheres, and grown in the presence of XMD8-92, a small-molecule inhibitor of ERK543 (Fig. 2a, b).

Self-renewal was then measured according to second-generation sphere formation without any additional treatment. Consistent with our hypothesis, XMD8-92 significantly reduced the frequency

of primary and secondary tumorsphere formation (_p_ < 0.05) (Fig. 2c). This was further associated with the disruption of sphere morphology and size (_p_ < 0.05) (Fig. 2b, d), with

minimal effects on cell viability (Supplementary Figure S2), suggesting that besides self-renewal, ERK5 inhibition also impairs the proliferative potential of stem-like malignant cells.

Worthy of note, sphere growth was conducted at clonal density (0.25-0.5 cells/μL) to avoid cell aggregation and sphere fusion. Single-cell assays confirmed the clonal origin of

tumorspheres44, as well as the ability of XMD8-92 to inhibit self-renewal and the rate of sphere formation in both HCT116 and SW620 cells (_p_ < 0.05) (Fig. 2e). Finally, to verify the

contribution of ERK5 to tumorsphere formation, ERK5 expression was specifically silenced by RNA interference in non-adherent HCT116 cultures (Fig. 2f, g). Interestingly, knockdown of ERK5

led to a marked decrease in the number (_p_ < 0.01) and size of HCT116-derived spheres (_p_ < 0.001) (Fig. 2h), phenocopying the effects of XMD8-92 treatment. These results demonstrate

that ERK5 inhibition depletes the population of sphere-initiating, self-renewing cells in colon cancer cultures. To investigate the molecular basis underlying the differential frequencies

of tumorsphere formation, the expression of core pluripotency transcription factors was next examined. ERK5 inhibition by XMD8-92 resulted in a significant downregulation of _SOX2_, _OCT4_,

and _NANOG_ in all cellular models under sphere-forming conditions, as assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR) (_p_ < 0.05) (Fig. 3a). These

results were further confirmed by immunoblot analysis in HCT116-derived tumorspheres (Fig. 3b). Similarly, flow cytometry analysis of aldehyde dehydrogenase (ALDH) activity, a

well-characterized marker of colon CSC subpopulations45, demonstrated a decrease in the proportion of ALDH-positive cells upon XMD8-92 treatment (_p_ < 0.05) (Fig. 3c). Taken together,

the aforementioned data demonstrate that ERK5 signaling inhibition suppresses malignant stem-like phenotypes and function, and support the notion that MEK5/ERK5 is required for sustained

stemness in colon cancer cells. ERK5 PHARMACOLOGICAL INHIBITION SENSITIZES COLON CANCER STEM-LIKE CELLS TO CHEMOTHERAPY Colon CSCs have been demonstrated to be highly resistant to

standard-of-care chemotherapy7,8,11, and combination strategies leading to the suppression of these therapy refractory cells may ultimately translate into improved treatment efficacy and

patient outcome. To evaluate the effect of ERK5 inhibition on cancer stem-like cell response to 5-fluorouracil (5-FU)-based chemotherapy, fully formed HCT116-derived tumorspheres were

treated with FOLFOX (5-FU plus oxaliplatin) or FOLFIRI (5-FU plus irinotecan), alone or in combination with XMD8-92 (Fig. 4a). Remarkably, XMD8-92-treated tumorspheres showed enhanced

sensitivity toward conventional FOLFOX and FOLFIRI treatment, as evidenced by an increase in cell death, compared to chemotherapy alone (_p_ < 0.01) (Fig. 4b). Consistent with these

observations, the combination of FOLFOX or FOLFIRI with XMD8-92 was further associated with increased caspase-3/7 activity (_p_ < 0.01) (Fig. 4c), PARP cleavage (_p_ < 0.05) (Fig. 4d,

left panel), and XIAP degradation (_p_ < 0.01) (Fig. 4d, right panel), demonstrating that ERK5 inhibition primes stem-like malignant populations to chemotherapy-induced apoptosis. ERK5

INHIBITION SUPPRESSES INTERLEUKIN-8 EXPRESSION THROUGH AN NUCLEAR FACTOR-KB-DEPENDENT MECHANISM To identify mechanisms downstream of MEK5/ERK5 that might contribute to stem-like cell

maintenance in colon cancer, we performed a comparative PCR array analysis of genes associated with CSC features in HCT116 tumorspheres treated with XMD8-92 or vehicle control. A total of 13

genes were found to be differentially expressed in response to ERK5 inhibition (log2-transformed fold change below −1 or above 1) (Fig. 5a, b). In line with the functional and biochemical

characterization of tumorspheres, XMD8-92 treatment led to an upregulation of the differentiation factor _GATA3_, and a downregulation of the pluripotency factors _KLF4_ and _MYC_, and CSC

markers _PROM1_/_CD133_ and _PLAUR_/_CD87_. This was further associated with a decrease in the expression of the ATP-binding cassette transporter _ABCG2_. On the other hand, inconsistent

effects were found for proliferation and migration-related genes (_KITLG_, _LIN28A_, _KLF17_, and _ZEB1_). Array results were validated by independent quantitative RT-PCR of a selection of

differentially expressed transcripts (Supplementary Figure S3). Apart from the impact of ERK5 inhibition on CSC-associated markers, gene expression profiling also revealed _NFKB1_ and the

nuclear factor-κB (NF-κB)-regulated _CXCL8/IL-8_46 as being downregulated in XMD8-92-treated tumorspheres (Fig. 5a, b). Quantitative RT-PCR confirmed that treatment with XMD8-92 reduced

_IL-8_ expression in HCT116, as well as SW480 and SW620 tumorspheres (_p_ < 0.01) (Fig. 5c). Similar results were observed when genetically silencing ERK5 in HCT116 cells under

sphere-forming conditions (_p_ < 0.01) (Fig. 5d). These data suggest that elimination of tumor cell populations with stem-like traits through ERK5 inhibition might be a result of

downstream _IL-8_ repression. Conversely, _IL-8_ mRNA levels were enriched in tumorsphere models where MEK5/ERK5 signaling was found to be induced (_p_ < 0.05) (Fig. 5e), and in SW480

cells expressing CA-MEK5 (_p_ < 0.05) (Fig. 5f), supporting the existence of a functional link between ERK5 activation and interleukin (IL)-8 signaling. Inhibition of ERK5 has been

previously shown to suppress IκB phosphorylation, preventing its degradation and subsequent NF-κB activation33. Here we investigated the relevance of the interplay between ERK5 and NF-κB

signaling pathways in colon CSC. Consistent with previous observations in monolayer-cultured cells36, XMD8-92 treatment in HCT116-derived tumorspheres resulted in decreased IκB

phosphorylation, and increased IκB protein levels (_p_ < 0.01) (Fig. 6a). Moreover, using a luciferase reporter system, NF-κB transcriptional activity was found to be significantly

impaired following XMD8-92 exposure (_p_ < 0.05) (Fig. 6b), mirroring the repression of _IL-8_ upon EKR5 inhibition, and suggesting a possible autocrine ERK5/NF-κB/IL-8 axis driving

stem-like cell malignancy. To investigate this further, NF-κB p65 and a dominant-negative-IκBα mutant (DN-IκBα) were respectively used to induce and block NF-κB activity in HCT116 cells

(Fig. 6c). Overexpression of NF-κB p65 led to a marked upregulation of _IL-8_, compared to empty vector cells, an outcome that was largely reversed by the addition of XMD8-92 (_p_ < 0.05)

(Fig. 6d). Conversely, DN-IκBα reduced _IL-8_ mRNA levels and abolished the effect of XMD8-92 in the expression of this chemokine. Overall, these data demonstrate that NF-κB is involved in

the regulation of IL-8 by ERK5, and provide a functional mechanism by which MEK5/ERK5 signaling contributes to the maintenance of stem-like properties in colon cancer. DISCUSSION In cancers

such as those of the colon, tumor initiation and progression occurs through aberrant activation and/or mutation of the same molecular mechanisms that control normal stem cell dynamics6,

within a network that goes beyond core pluripotency pathways, and is currently recognized to be controlled by multiple protein kinase cascades47. Exemplifying this phenomenon is the

MEK5/ERK5 signaling pathway, which has been shown to participate in both development and tumorigenesis16. In this framework, we hypothesized that ERK5-mediated signaling could contribute to

the maintenance of a stem-like population in colon cancer. Particularly, we demonstrate that MEK5/ERK5 activation is increased in several cell line-derived models enriched for malignant stem

cells (Fig. 1); and that ERK5 inhibition using XMD8-92 suppresses both self-renewal and the expression of colon CSC-associated markers (Figs. 2 and 3). In line with our results, ERK5 has

been previously identified as a critical player for sphere formation and tumor initiation in lung carcinoma cells48. Additionally, the suggested role of ERK5 in defining a CSC-like phenotype

is consistent with the notion that activation of epithelial-to-mesenchymal transition (EMT) programs induces the acquisition of CSC traits that facilitate metastasis formation49. In this

regard, we have previously demonstrated that MEK5/ERK5 activation is associated with upregulation of the mesenchymal marker vimentin, promoting colon cancer cell invasive and metastatic

behavior in an orthotopic xenograft model33. Moreover, several other studies reported that MEK5 and ERK5 regulate EMT features, generation of circulating tumor cells, and metastatic seeding

in different tumor contexts50,51,52. Still, further investigation is required to evaluate the possible influence of ERK5-mediated signaling to the molecular mechanisms underlying the

connection between EMT and the CSC state. Apart from the role of malignant stem-like cells in tumor initiation and metastasis, the CSC concept also provides a framework to understand therapy

resistance6. Evidence from patient-derived three-dimensional cultures and xenograft models indicate that colon CSCs are intrinsically drug-resistant8,9,10,11. Moreover, the proportion of

cells expressing CSC-associated markers was found enriched within residual tumors of colon cancer patients receiving chemoradiotherapy7. Therefore, identification of targetable signaling

pathways controlling colon cancer stem-like phenotypes will undoubtedly fuel the development of combination regimens to overcome current therapy limitations. We have recently shown that ERK5

inhibition enhances the anticancer properties of 5-FU in a murine xenograft model of colon cancer36. Here we extend these earlier studies by revealing that XMD8-92 treatment sensitizes

HCT116 cancer stem-like cells to 5-FU-based chemotherapy (Fig. 4), establishing a novel strategy to eliminate drug-resistant populations generated upon phenotypic switching into the CSC

state. In parallel, we also found that ERK5 inhibition in tumorspheres leads to downregulation of _ABCG2_ (Fig. 5 and Supplementary Figure S3), a drug-efflux pump that is responsible for

acquired resistance to both 5-FU53 and irinotecan54, also contributing to CSC malignant properties55. Indeed, and in agreement with our results, the ERK5/MEF2 pathway has been proposed to

regulate the expression of several ABC transporters, among which _ABCG2_56. It is therefore possible that part of the mechanism behind the increased susceptibility of stem-like colon cancer

cells to chemotherapy upon ERK5 inhibition could involve _ABCG2_ repression. Finally, our data demonstrate that NF-κB-mediated _IL-8_ expression might be a fundamental element of CSC-like

function downstream of MEK5/ERK5 signaling (Figs. 5 and 6). In colon cancer, aberrant expression of the CXC chemokine IL-8, in tumor tissues or in circulation, was shown to be associated

with poor differentiation, depth of invasion, and distant metastasis57,58,59. Functionally, IL-8 signaling promotes EMT, stem cell-like traits, and chemoresistance60,61,62. Regarding CSCs,

the pro-inflammatory and angiogenic activity of IL-8 is known to be essential for the establishment of a supportive microenvironment for self-renewal and stem-like cell survival63. However,

according to our experimental conditions, we suggest that IL-8 expression by tumor cells might also contribute to sustained stemness through autocrine signaling. Indeed, a similar feedback

loop mechanism has already been proposed for the regulation of colon cancer cell proliferation and migration60,64. Strengthening our hypothesis, while NF-κB controls IL-8 expression46, this

chemokine is in turn responsible for triggering NF-κB transcriptional activity65. Consistently, NF-κB signaling has also been linked to CSC-like features in several solid tumors, including

glioblastoma, breast, prostate, and non-small cell lung cancer66,67,68,69. On the other hand, pharmacological inhibition and genetic knockdown of either MEK5 or ERK5 were reported to

suppress lipopolysaccharide-, IL-1β-, and tumor necrosis factor-α-induced production of IL-8 in primary human endothelial cells and monocytes70. Similarly, we demonstrate that specifically

silencing ERK5 recapitulates the effects of XMD8-92-mediated inhibition of ERK5 kinase activity, depleting the population of sphere-initiating cells (Fig. 2), and the expression of _IL-8_ in

HCT116 tumorspheres (Fig. 5). Nevertheless, we cannot fully exclude that putative XMD8-92 off-target activity may partially account for the observed phenotypic effects of this

small-molecule inhibitor against colon cancer stem-like cells71,72. Moreover, although cancer cell lines are representative of the different colorectal cancer molecular subtypes, which

validates their utility as tools to investigate tumor biology and drug response73,74, future studies will be necessary to evaluate the impact of ERK5 inhibition on patient-derived in vitro

and in vivo models. Taken together, our findings provide proof of principle that pharmacological inhibition of ERK5 may be an effective strategy to target self-renewing, drug-resistant colon

cancer stem-like cells. Adding to the clinical relevance of this signaling pathway, aberrant MEK5/ERK5 activation also contributes to increased tumor cell proliferation and metastasis33,

inducing a chemoresistant phenotype in both CSCs and non-CSCs36. The plurality of mechanisms through which ERK5 activity drives the process of tumorigenesis reinforces the therapeutic

potential of blocking this cascade in colon cancer treatment. Still, heterogeneous tumor masses comprising different populations of differentiated and cancer stem-like cells are expected to

be sustained by different oncogenic mechanisms. Therefore, the introduction of ERK5-targeting agents in clinical evaluation should be envisioned as part of combination regimens designed to

avoid resistance and tumor recurrence, bringing together conventional cytotoxic drugs and innovative targeted therapies. MATERIALS AND METHODS CELL CULTURE Human HCT116, HT29, SW480, and

SW620 colorectal carcinoma cell lines were obtained from ECACC (Porton Down, UK), passaged for <6 months after resuscitation, and routinely tested for mycoplasma contamination using

Mycoalert detection kit (Lonza, Basel, Switzerland). Cells were cultured in adherent conditions in McCoy’s 5A (HCT116), RPMI 1640 (HT29), or Dulbecco’s modified Eagle’s medium (DMEM) (SW480

and SW620), all supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution (all from Gibco, Thermo Fisher Scientific, Paisley, UK). For the

generation of tumorspheres, cells were grown in non-adherent conditions in serum-free DMEM/F12 medium containing 2% B27 supplement, 1% N2 supplement, 1% non-essential amino acids, 1% sodium

pyruvate, 1% penicillin-streptavidin, 4 μg/mL heparin, 40 ng/mL recombinant human epidermal growth factor (all from Gibco) and 20 ng/mL recombinant human basic fibroblast growth factor

(Peprotech, London, UK). All cell cultures were maintained at 37 °C under a humidified atmosphere of 5% CO2. SMALL MOLECULES AND CHEMOTHERAPEUTIC AGENTS The ERK5 pharmacological inhibitor

XMD8-92 was obtained from Selleckchem (Madrid, Spain) and prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich, MO, USA). Clinical-grade 5-FU, oxaliplatin, and irinotecan were kindly provided

by Hospital São Francisco Xavier (Lisbon, Portugal), and diluted to stock concentrations in phosphate-buffered saline (Gibco, Thermo Fisher Scientific). Stock solutions were aliquoted and

stored at −80 and −20 °C, respectively. All subsequent dilutions were freshly prepared in culture medium. Experiments were performed in parallel with DMSO vehicle control. Final DMSO

concentration was always 0.1%. CELL TRANSFECTION For overexpression experiments, CA-MEK5 plasmid (pWPI-MEK5DD; S313D/T317D) was kindly provided by Dr. Robert C. Doebele (University of

Colorado, CO, USA)75. Constructs for NF-κB p65 (pCMV4 p65)76 and DN-IκBα (pCMX IkB alpha M; S32A/S36A)77 were obtained from Addgene (#21966 and #12329, respectively). For small interfering

RNA (siRNA)-mediated knockdown of ERK5, the MAPK7 Silencer Select was used (#s11149; Applied Biosystems, Thermo Fisher Scientific). HCT116 and SW480 cells were plated at 3 × 105 cells/well

on 35 mm dishes and transfected with either 1 μg of plasmid DNA or 80 nM of siRNA using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific), according to the manufacturer’s

instructions. In both cases, cells were allowed to grow for at least 24 h before further treatment or re-plating for tumorsphere formation. SPHERE-FORMING ASSAY To measure tumorsphere

formation, colon cancer cells were plated as single cells in 24-well ultra-low attachment plates (Corning, NY, USA) at 250–500 cells/well, and cultured in 1 mL serum-free DMEM/F12

supplemented with growth factors. After 8 days, spheres were collected, dissociated into single cells, and reseeded as above for secondary sphere formation. For each generation, the number

of tumorspheres was determined under an inverted microscope. Additionally, the number of cells per tumorsphere was quantified using trypan blue exclusion assay. Alternatively, cells were

sorted at a density of 1 cell/well into 96-well ultra-low attachment plates (BD FACS Aria III, BD Biosciences, CA, USA), and allowed to grow in 200 μL tumorsphere medium. The wells without

cells were excluded from analysis one day after plating, and a minimum of 90 wells per condition was considered. Sphere-forming efficiency was calculated after 14 days according to the

proportion of wells with tumorspheres versus initially seeded wells. In all cases, cells were allowed to adapt for 24 h and then treated with 4 μM XMD8-92 or DMSO vehicle control, except for

second-generation spheres, which were grown without further treatment. ALDEFLUOR ASSAY Cells with high ALDH enzymatic activity were identified using the Aldefluor assay (StemCell

Technologies, Grenoble, France) according to the manufacturer’s protocol. In brief, 5,000–10,000 single cells were seeded in 5 mL tumorsphere medium using non-tissue culture-treated 55 mm

dishes (Gosselin, Hazebrouck, France), cultured for 24 h, and then treated with either 4 μM XMD8-92 or DMSO vehicle control. Eight-day tumorspheres were collected, dissociated into single

cells, resuspended in assay buffer containing 1.5 μM BODIPY-aminoacetaldehyde, and incubated for 40 min at 37 °C. Diethylaminobenzaldehyde (15 μM), a specific ALDH inhibitor, was used as a

negative control for each reaction. Samples were then centrifuged, resuspended in fresh assay buffer, and stored on ice until flow cytometric analysis. Sample acquisition was performed in a

BD LSRFortessa Cell Analyzer cytometer (BD Biosciences). A total of 10,000 cells were analyzed for each test and control sample pair, and the percentage of ALDHhigh cells was determined

using FlowJo software (version 10.0.7; Tree Star, CA, USA). CELL DEATH AND CASPASE ACTIVITY ASSAYS HCT116 cells were plated in 24-well ultra-low attachment plates and stem cell medium at a

density of 500 cells/well. Resulting 8-day tumorsphere cultures were treated for 3 days with FOLFOX (1.25 μM oxaliplatin plus 50 μM 5-FU), or FOLFIRI (1 μM irinotecan plus 50 μM 5-FU)

chemotherapeutic regimens78, alone or in combination with 4 μM XMD8-92. The in vitro cytotoxic effect of chemotherapy was evaluated using ToxiLight bioassay kit (Lonza) to measure the amount

of adenylate kinase (AK) released from plasma membrane-damaged cells into tumorsphere supernatants, following the manufacturer’s instructions. Further, the activity of effector caspases-3

and -7 was measured using Caspase-Glo 3/7 Assay (Promega, WI, USA). For this purpose, tumorspheres were collected at 300 × _g_ for 7 min, dissociated into single cells, and resuspended in

fresh growth medium. Cell suspensions were then mixed with an equal volume of Caspase-Glo 3/7 reagent, and incubated for 30 min at room temperature, protected from light. Resulting

luminescence was measured using the GloMax-Multi+ Detection System (Promega). Experimental AK release and caspase-3/-7 activity levels were normalized by the number of cells per well.

QUANTITATIVE RT-PCR For gene expression analysis, tumorspheres were grown and treated as described for Aldefluor activity evaluation. Additionally, comparative studies were conducted by

culturing colon cancer cells as monolayers in traditional medium supplemented with FBS. Total RNA was extracted using Ribozol (VWR International, PA, USA), treated with RNase-free

recombinant DNase I (Roche, Mannheim, Germany), and reverse-transcribed to complementary DNA using the NZY First-Strand cDNA Synthesis Kit (NZYTech, Lisbon, Portugal), all according to the

manufacturers’ instructions. Quantitative real-time PCR was performed in 5 μL duplicate reactions on a 384-well QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, Thermo Fisher

Scientific), using the SensiFAST SYBR Hi-ROX kit (Bioline, London, UK), following manufacturer’s protocol. Primer sequences are listed in Supplementary Table S1. For each sample,

quantification of gene expression was performed using the relative standard curve method and normalized to _ACTB_ levels. TOTAL PROTEIN ISOLATION AND IMMUNOBLOTTING Total protein extraction

and immunoblot analysis were performed as previously described36. Briefly, 40 μg of total protein extracts were denatured, separated on 8 or 10% sodium dodecyl sulfate polyacrylamide

electrophoresis gels, and transferred onto nitrocellulose membranes. Steady-state protein levels were evaluated using primary rabbit antibodies reactive to ERK5 (#3372), OCT4 (#2750; Cell

Signaling Technology, MA, USA), SOX2 (#AB5603, Merck Millipore, MA, USA), p-MEK5 (#sc-135702), PARP (#sc-7150), NF-κB (#sc-372), IκBα (#sc-371), or XIAP (#sc-11426; Santa Cruz Biotechnology,

CA, USA); or primary mouse antibodies against MEK5 (#sc-135986), NANOG (#sc-134218, Santa Cruz Biotechnology), or p-IκBα (#9246; Cell Signaling Technology). β-actin (#A5541; Sigma-Aldrich)

and GAPDH (#sc-32233) were used as loading controls. Following incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories, CA, USA), the 

proteins of interest were detected by chemiluminescence using SuperSignal reagents (Pierce, Thermo Fisher Scientific), on a ChemiDoc XRS+ imaging system (Bio-Rad). Densitometric analysis was

performed using the Image Lab software (version 5.1; Bio-Rad). GENE EXPRESSION PROFILING Differential gene expression between DMSO- and XMD8-92-treated tumorspheres was evaluated using the

Human Cancer Stem Cells RT2 Profiler PCR Array (PAHS-176Z; Qiagen, MD, USA) according to the manufacturer’s instructions. For each condition, pools were obtained by combining equal amounts

of total RNA from five different experiments. Complementary DNA synthesis was performed with 800 ng of DNase I-treated RNA (Roche) and the RT2 First Strand Kit (Qiagen). Real-time PCR was

run on a 384-well QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific), using the RT2 SYBR Green ROX qPCR master mix (Qiagen). Duplicate reactions for all genes, as well as

quality controls for genomic DNA contamination, reverse transcription efficiency, and PCR array reproducibility were included. Data analysis was performed using the GeneGlobe online platform

(https://www.qiagen.com/geneglobe/). Relative gene expression over control samples was determined as per the comparative cycle threshold (ΔΔCt) method and normalized to the geometric mean

of _B2M_ and _HPRT_ reference genes (ΔCt = Ctreference − Cttarget; ΔΔCt = ΔCtXMD8-92 − ΔCtDMSO). A cutoff value of log2-fold change (ΔΔCt) ≥ 1 was defined for the selection of differentially

expressed transcripts. Genes with Ct values above 34 or standard deviations between technical replicates superior to 0.5 were excluded from analysis. Results for each detectable gene are

shown in Supplementary Table S2. NF-ΚB LUCIFERASE REPORTER ASSAY NF-κB transcriptional activity was measured using the Cignal NF-κB Pathway Reporter Assay Kit (Qiagen), according to the

manufacturer’s specifications. Briefly, HCT116 cells were seeded at 2 × 104 cells/well on 96-well plates and transfected with 100 ng of luciferase construct harboring NF-κB response elements

using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific). Non-inducible and constitutively expressed firefly luciferase constructs were used as negative and positive controls,

respectively. A constitutive Renilla luciferase vector was included in all mixes (40:1) to normalize transfection efficiency and monitor cell viability. Sixteen hours post transfection,

cells were treated with 4 μM XMD8-92 or DMSO vehicle control. Luciferase activities were assayed 8 h after treatment using the Dual-Luciferase Reporter Assay System (Promega). STATISTICAL

ANALYSIS All data are expressed as mean ± standard error of the mean from at least three independent experiments. Statistical significances were determined using unpaired two-tailed

Student’s _t_-test. Values of _p_ < 0.05 were considered statistically significant. REFERENCES * Shackleton, M., Quintana, E., Fearon, E. R. & Morrison, S. J. Heterogeneity in cancer:

cancer stem cells versus clonal evolution. _Cell_ 138, 822–829 (2009). Article  CAS  Google Scholar  * Ricci-Vitiani, L. et al. Identification and expansion of human colon-cancer-initiating

cells. _Nature_ 445, 111–115 (2007). Article  CAS  Google Scholar  * O’Brien, C. A., Pollett, A., Gallinger, S. & Dick, J. E. A human colon cancer cell capable of initiating tumour

growth in immunodeficient mice. _Nature_ 445, 106–110 (2007). Article  Google Scholar  * Dalerba, P. et al. Phenotypic characterization of human colorectal cancer stem cells. _Proc. Natl

Acad. Sci. USA_ 104, 10158–10163 (2007). Article  CAS  Google Scholar  * Clarke, M. F. et al. Cancer stem cells–perspectives on current status and future directions: AACR Workshop on cancer

stem cells. _Cancer Res._ 66, 9339–9344 (2006). Article  CAS  Google Scholar  * Zeuner, A., Todaro, M., Stassi, G. & De Maria, R. Colorectal cancer stem cells: from the crypt to the

clinic. _Cell Stem Cell_ 15, 692–705 (2014). Article  CAS  Google Scholar  * Wilson, B. J. et al. ABCB5 identifies a therapy-refractory tumor cell population in colorectal cancer patients.

_Cancer Res._ 71, 5307–5316 (2011). Article  CAS  Google Scholar  * Todaro, M. et al. Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. _Cell

Stem Cell_ 1, 389–402 (2007). Article  CAS  Google Scholar  * Dylla, S. J. et al. Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy. _PLoS ONE_ 3, e2428

(2008). Article  Google Scholar  * Hoey, T. et al. DLL4 blockade inhibits tumor growth and reduces tumor-initiating cell frequency. _Cell Stem Cell_ 5, 168–177 (2009). Article  CAS  Google

Scholar  * Colak, S. et al. Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells. _Cell Death Differ._ 21, 1170–1177 (2014). Article  CAS  Google Scholar  *

Merlos-Suarez, A. et al. The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. _Cell Stem Cell_ 8, 511–524 (2011). Article  CAS  Google

Scholar  * de Sousa, E. M. F. et al. Methylation of cancer-stem-cell-associated Wnt target genes predicts poor prognosis in colorectal cancer patients. _Cell Stem Cell_ 9, 476–485 (2011).

Article  Google Scholar  * Dalerba, P. et al. CDX2 as a prognostic biomarker in stage II and stage III colon cancer. _N. Engl. J. Med._ 374, 211–222 (2016). Article  CAS  Google Scholar  *

Karamboulas, C. & Ailles, L. Developmental signaling pathways in cancer stem cells of solid tumors. _Biochim. Biophys. Acta_ 1830, 2481–2495 (2013). Article  CAS  Google Scholar  *

Nithianandarajah-Jones, G. N., Wilm, B., Goldring, C. E., Muller, J. & Cross, M. J. ERK5: structure, regulation and function. _Cell. Signal._ 24, 2187–2196 (2012). Article  CAS  Google

Scholar  * Sohn, S. J., Sarvis, B. K., Cado, D. & Winoto, A. ERK5 MAPK regulates embryonic angiogenesis and acts as a hypoxia-sensitive repressor of vascular endothelial growth factor

expression. _J. Biol. Chem._ 277, 43344–43351 (2002). Article  CAS  Google Scholar  * Regan, C. P. et al. Erk5 null mice display multiple extraembryonic vascular and embryonic cardiovascular

defects. _Proc. Natl Acad. Sci. USA_ 99, 9248–9253 (2002). Article  CAS  Google Scholar  * Yan, L. et al. Knockout of ERK5 causes multiple defects in placental and embryonic development.

_BMC Dev. Biol._ 3, 11 (2003). Article  Google Scholar  * Wang, X. et al. Targeted deletion of mek5 causes early embryonic death and defects in the extracellular signal-regulated kinase

5/myocyte enhancer factor 2 cell survival pathway. _Mol. Cell. Biol._ 25, 336–345 (2005). Article  Google Scholar  * Liu, L. et al. Extracellular signal-regulated kinase (ERK) 5 is necessary

and sufficient to specify cortical neuronal fate. _Proc. Natl Acad. Sci. USA_ 103, 9697–9702 (2006). Article  CAS  Google Scholar  * Li, T. et al. Targeted deletion of the ERK5 MAP kinase

impairs neuronal differentiation, migration, and survival during adult neurogenesis in the olfactory bulb. _PLoS ONE_ 8, e61948 (2013). Article  CAS  Google Scholar  * Wang, W. et al.

Genetic activation of ERK5 MAP kinase enhances adult neurogenesis and extends hippocampus-dependent long-term memory. _J. Neurosci._ 34, 2130–2147 (2014). Article  CAS  Google Scholar  *

Wang, W. et al. Inducible activation of ERK5 MAP kinase enhances adult neurogenesis in the olfactory bulb and improves olfactory function. _J. Neurosci._ 35, 7833–7849 (2015). Article  CAS 

Google Scholar  * Dinev, D. et al. Extracellular signal regulated kinase 5 (ERK5) is required for the differentiation of muscle cells. _EMBO Rep._ 2, 829–834 (2001). Article  CAS  Google

Scholar  * Sunadome, K. et al. ERK5 regulates muscle cell fusion through Klf transcription factors. _Dev. Cell_ 20, 192–205 (2011). Article  CAS  Google Scholar  * Sohn, S. J., Lewis, G. M.

& Winoto, A. Non-redundant function of the MEK5-ERK5 pathway in thymocyte apoptosis. _EMBO J._ 27, 1896–1906 (2008). Article  CAS  Google Scholar  * Wang, X. et al. The MAPK ERK5, but

not ERK1/2, inhibits the progression of monocytic phenotype to the functioning macrophage. _Exp. Cell Res._ 330, 199–211 (2015). Article  CAS  Google Scholar  * Giurisato, E. et al. Myeloid

ERK5 deficiency suppresses tumor growth by blocking protumor macrophage polarization via STAT3 inhibition. _Proc. Natl Acad. Sci. USA_ 115, E2801–E2810 (2018). Article  CAS  Google Scholar 

* Williams, C. A. et al. Erk5 is a key regulator of naive-primed transition and embryonic stem cell identity. _Cell Rep._ 16, 1820–1828 (2016). Article  CAS  Google Scholar  * de Jong, P. R.

et al. ERK5 signalling rescues intestinal epithelial turnover and tumour cell proliferation upon ERK1/2 abrogation. _Nat. Commun._ 7, 11551 (2016). Article  Google Scholar  * Osaki, L. H.

& Gama, P. MAPKs and signal transduction in the control of gastrointestinal epithelial cell proliferation and differentiation. _Int. J. Mol. Sci._ 14, 10143–10161 (2013). Article  Google

Scholar  * Simoes, A. E. et al. Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF-kappaB activation. _Cell Death Dis._ 6, e1718 (2015). Article  CAS  Google

Scholar  * Hu, B. et al. Expression of the phosphorylated MEK5 protein is associated with TNM staging of colorectal cancer. _BMC Cancer_ 12, 127 (2012). Article  CAS  Google Scholar  *

Diao, D. et al. MEK5 overexpression is associated with the occurrence and development of colorectal cancer. _BMC Cancer_ 16, 302 (2016). Article  Google Scholar  * Pereira, D. M. et al.

MEK5/ERK5 signaling inhibition increases colon cancer cell sensitivity to 5-fluorouracil through a p53-dependent mechanism. _Oncotarget_ 7, 34322–34340 (2016). PubMed  PubMed Central  Google

Scholar  * Simoes, A. E., Rodrigues, C. M. & Borralho, P. M. The MEK5/ERK5 signalling pathway in cancer: a promising novel therapeutic target. _Drug Discov. Today_ 21, 1654–1663 (2016).

Article  CAS  Google Scholar  * Weiswald, L. B., Bellet, D. & Dangles-Marie, V. Spherical cancer models in tumor biology. _Neoplasia_ 17, 1–15 (2015). Article  Google Scholar  *

Bielecka, Z. F., Maliszewska-Olejniczak, K., Safir, I. J., Szczylik, C. & Czarnecka, A. M. Three-dimensional cell culture model utilization in cancer stem cell research. _Biol. Rev.

Camb. Philos. Soc._ 92, 1505–1520 (2017). Article  Google Scholar  * Kanwar, S. S., Yu, Y., Nautiyal, J., Patel, B. B. & Majumdar, A. P. The Wnt/beta-catenin pathway regulates growth and

maintenance of colonospheres. _Mol. Cancer_ 9, 212 (2010). Article  Google Scholar  * Bitarte, N. et al. MicroRNA-451 is involved in the self-renewal, tumorigenicity, and chemoresistance of

colorectal cancer stem cells. _Stem Cells_ 29, 1661–1671 (2011). Article  CAS  Google Scholar  * Prabhu, V. V. et al. Small-molecule prodigiosin restores p53 tumor suppressor activity in

chemoresistant colorectal cancer stem cells via c-Jun-mediated DeltaNp73 inhibition and p73 activation. _Cancer Res._ 76, 1989–1999 (2016). Article  CAS  Google Scholar  * Yang, Q. et al.

Pharmacological inhibition of BMK1 suppresses tumor growth through promyelocytic leukemia protein. _Cancer Cell_ 18, 258–267 (2010). Article  CAS  Google Scholar  * Pastrana, E.,

Silva-Vargas, V. & Doetsch, F. Eyes wide open: a critical review of sphere-formation as an assay for stem cells. _Cell Stem Cell_ 8, 486–498 (2011). Article  CAS  Google Scholar  *

Huang, E. H. et al. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. _Cancer Res._ 69,

3382–3389 (2009). Article  CAS  Google Scholar  * Mukaida, N., Okamoto, S., Ishikawa, Y. & Matsushima, K. Molecular mechanism of interleukin-8 gene expression. _J. Leukoc. Biol._ 56,

554–558 (1994). Article  CAS  Google Scholar  * Fernandez-Alonso, R., Bustos, F., Williams, C. A. C. & Findlay, G. M. Protein kinases in pluripotency-beyond the usual suspects. _J. Mol.

Biol._ 429, 1504–1520 (2017). Article  CAS  Google Scholar  * Song, C. et al. Inhibition of BMK1 pathway suppresses cancer stem cells through BNIP3 and BNIP3L. _Oncotarget_ 6, 33279–33289

(2015). PubMed  PubMed Central  Google Scholar  * Shibue, T. & Weinberg, R. A. EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. _Nat. Rev. Clin. Oncol._

14, 611–629 (2017). Article  Google Scholar  * Mehta, P. B. et al. MEK5 overexpression is associated with metastatic prostate cancer, and stimulates proliferation, MMP-9 expression and

invasion. _Oncogene_ 22, 1381–1389 (2003). Article  CAS  Google Scholar  * Javaid, S. et al. MAPK7 regulates EMT features and modulates the generation of CTCs. _Mol. Cancer Res._ 13, 934–943

(2015). Article  CAS  Google Scholar  * Pavan, S. et al. A kinome-wide high-content siRNA screen identifies MEK5-ERK5 signaling as critical for breast cancer cell EMT and metastasis.

_Oncogene_ 37, 4197–4213 (2018). Article  CAS  Google Scholar  * Yuan, J. et al. Role of BCRP as a biomarker for predicting resistance to 5-fluorouracil in breast cancer. _Cancer Chemother.

Pharmacol._ 63, 1103–1110 (2009). Article  CAS  Google Scholar  * Tuy, H. D. et al. ABCG2 expression in colorectal adenocarcinomas may predict resistance to irinotecan. _Oncol. Lett._ 12,

2752–2760 (2016). Article  CAS  Google Scholar  * Fang, D. D. et al. Expansion of CD133(+) colon cancer cultures retaining stem cell properties to enable cancer stem cell target discovery.

_Br. J. Cancer_ 102, 1265–1275 (2010). Article  CAS  Google Scholar  * Belkahla, S. et al. Changes in metabolism affect expression of ABC transporters through ERK5 and depending on p53

status. _Oncotarget_ 9, 1114–1129 (2018). Article  Google Scholar  * Ueda, T., Shimada, E. & Urakawa, T. Serum levels of cytokines in patients with colorectal cancer: possible

involvement of interleukin-6 and interleukin-8 in hematogenous metastasis. _J. Gastroenterol._ 29, 423–429 (1994). Article  CAS  Google Scholar  * Terada, H., Urano, T. & Konno, H.

Association of interleukin-8 and plasminogen activator system in the progression of colorectal cancer. _Eur. Surg. Res._ 37, 166–172 (2005). Article  CAS  Google Scholar  * Cacev, T.,

Radosevic, S., Krizanac, S. & Kapitanovic, S. Influence of interleukin-8 and interleukin-10 on sporadic colon cancer development and progression. _Carcinogenesis_ 29, 1572–1580 (2008).

Article  CAS  Google Scholar  * Bates, R. C., DeLeo, M. J. 3rd & Mercurio, A. M. The epithelial-mesenchymal transition of colon carcinoma involves expression of IL-8 and CXCR-1-mediated

chemotaxis. _Exp. Cell Res._ 299, 315–324 (2004). Article  CAS  Google Scholar  * Ning, Y. et al. Interleukin-8 is associated with proliferation, migration, angiogenesis and chemosensitivity

in vitro and in vivo in colon cancer cell line models. _Int. J. Cancer_ 128, 2038–2049 (2011). Article  CAS  Google Scholar  * Hwang, W. L. et al. SNAIL regulates interleukin-8 expression,

stem cell-like activity, and tumorigenicity of human colorectal carcinoma cells. _Gastroenterology_ 141, 279–291 (2011). 291 e271-275. Article  CAS  Google Scholar  * Korkaya, H., Liu, S.

& Wicha, M. S. Regulation of cancer stem cells by cytokine networks: attacking cancer’s inflammatory roots. _Clin. Cancer Res._ 17, 6125–6129 (2011). Article  CAS  Google Scholar  *

Brew, R. et al. Interleukin-8 as an autocrine growth factor for human colon carcinoma cells in vitro. _Cytokine_ 12, 78–85 (2000). Article  CAS  Google Scholar  * Manna, S. K. & Ramesh,

G. T. Interleukin-8 induces nuclear transcription factor-kappaB through a TRAF6-dependent pathway. _J. Biol. Chem._ 280, 7010–7021 (2005). Article  CAS  Google Scholar  * Rajasekhar, V. K.,

Studer, L., Gerald, W., Socci, N. D. & Scher, H. I. Tumour-initiating stem-like cells in human prostate cancer exhibit increased NF-kappaB signalling. _Nat. Commun._ 2, 162 (2011).

Article  Google Scholar  * Liu, M. et al. The canonical NF-kappaB pathway governs mammary tumorigenesis in transgenic mice and tumor stem cell expansion. _Cancer Res._ 70, 10464–10473

(2010). Article  CAS  Google Scholar  * Garner, J. M. et al. Constitutive activation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappaB signaling in

glioblastoma cancer stem cells regulates the Notch pathway. _J. Biol. Chem._ 288, 26167–26176 (2013). Article  CAS  Google Scholar  * Zakaria, N., Mohd Yusoff, N., Zakaria, Z., Widera, D.

& Yahaya, B. H. Inhibition of NF-kappaB signaling reduces the stemness characteristics of lung cancer stem cells. _Front. Oncol._ 8, 166 (2018). Article  Google Scholar  * Wilhelmsen, K.

et al. Extracellular signal-regulated kinase 5 promotes acute cellular and systemic inflammation. _Sci. Signal._ 8, ra86 (2015). Article  Google Scholar  * Sureban, S. M. et al. XMD8-92

inhibits pancreatic tumor xenograft growth via a DCLK1-dependent mechanism. _Cancer Lett._ 351, 151–161 (2014). Article  CAS  Google Scholar  * Lin, E. C. et al. ERK5 kinase activity is

dispensable for cellular immune response and proliferation. _Proc. Natl Acad. Sci. USA_ 113, 11865–11870 (2016). Article  CAS  Google Scholar  * Schlicker, A. et al. Subtypes of primary

colorectal tumors correlate with response to targeted treatment in colorectal cell lines. _BMC Med. Genomics_ 5, 66 (2012). Article  CAS  Google Scholar  * Mouradov, D. et al. Colorectal

cancer cell lines are representative models of the main molecular subtypes of primary cancer. _Cancer Res._ 74, 3238–3247 (2014). Article  CAS  Google Scholar  * Doebele, R. C. et al. A

novel interplay between Epac/Rap1 and mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) regulates thrombospondin to control angiogenesis. _Blood_

114, 4592–4600 (2009). Article  CAS  Google Scholar  * Ballard, D. W. et al. The 65-kDa subunit of human NF-kappa B functions as a potent transcriptional activator and a target for

v-Rel-mediated repression. _Proc. Natl Acad. Sci. USA_ 89, 1875–1879 (1992). Article  CAS  Google Scholar  * Van Antwerp, D. J., Martin, S. J., Kafri, T., Green, D. R. & Verma, I. M.

Suppression of TNF-alpha-induced apoptosis by NF-kappaB. _Science_ 274, 787–789 (1996). Article  Google Scholar  * Wielenga, M. C. B. et al. ER-stress-induced differentiation sensitizes

colon cancer stem cells to chemotherapy. _Cell Rep._ 13, 489–494 (2015). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The authors thank Dr. Robert C. Doebele

(University of Colorado, CO, USA) for the kind gift of the pWPI-GFP expression construct encoding constitutively active MEK5. We also wish to thank Hospital São Francisco Xavier (Lisbon,

Portugal) for providing clinical grade 5-fluorouracil, oxaliplatin, and irinotecan. This work was supported by _Fundação para a Ciência e a Tecnologia_ (FCT) through fellowships

SFRH/BD/88619/2012 (S.E.G.) and SFRH/BD/96517/2013 (D.M.P.). This project received funding from European Structural & Investment Funds through the COMPETE Programme and from National

Funds through FCT under the Programme grant SAICTPAC/0019/2015. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy,

Universidade de Lisboa, Lisbon, Portugal Diane M. Pereira, Sofia. E. Gomes, Pedro M. Borralho & Cecília M. P. Rodrigues Authors * Diane M. Pereira View author publications You can also

search for this author inPubMed Google Scholar * Sofia. E. Gomes View author publications You can also search for this author inPubMed Google Scholar * Pedro M. Borralho View author

publications You can also search for this author inPubMed Google Scholar * Cecília M. P. Rodrigues View author publications You can also search for this author inPubMed Google Scholar

CORRESPONDING AUTHOR Correspondence to Cecília M. P. Rodrigues. ETHICS DECLARATIONS CONFLICT OF INTEREST The authors declare that they have no conflict of interest. ADDITIONAL INFORMATION

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ARTICLE Pereira, D.M., Gomes, S.E., Borralho, P.M. _et al._ MEK5/ERK5 activation regulates colon cancer stem-like cell properties. _Cell Death Discovery_ 5, 68 (2019).

https://doi.org/10.1038/s41420-019-0150-1 Download citation * Received: 30 October 2018 * Revised: 21 November 2018 * Accepted: 29 November 2018 * Published: 11 February 2019 * DOI:

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