The Endosomal Protein CEMIP Links WNT Signaling to MEK1-ERK1/2 Activation in Selumetinib-Resistant Intestinal Organoids

Abstract

Mitogen-activated protein kinase (MAPK) signaling pathways exhibit constitutive activity in colon cancer and are also implicated in promoting acquired resistance to MEK1 inhibition. This study demonstrates that BRAFV600E-mutated colorectal cancers develop resistance to MEK1 inhibition through the induction of CEMIP, a scaffold protein, via a pathway dependent on beta-catenin and FRA-1. CEMIP was localized within endosomes and found to bind to MEK1, thereby sustaining ERK1/2 activation in BRAFV600E-mutated colorectal cancers that had become resistant to MEK1 inhibitors. This CEMIP-dependent pathway maintained the protein levels of c-Myc through ERK1/2, conferring a metabolic advantage to the resistant cells, potentially by supporting amino acid synthesis. Silencing CEMIP effectively circumvented resistance to MEK1 inhibition, partially through a reduction in both ERK1/2 signaling and c-Myc levels. Collectively, these findings identify a cross-talk mechanism between the Wnt and MAPK signaling cascades, involving CEMIP. Activation of this pathway promotes survival, possibly by regulating the levels of specific amino acids via a Myc-associated cascade. Targeting this node may represent a promising therapeutic strategy for treating colon cancers that have developed resistance to targeted therapies.

Precis

Colorectal cancer that has become resistant to MEK1 inhibitors relies on CEMIP, a scaffold and endosomal protein, to maintain ERK1/2 signaling and Myc-driven transcription.

Introduction

Colorectal cancer (CRC) stands as the second leading cause of cancer-related deaths in Western countries, arising from a complex interplay of genetic alterations that result in the persistent activation of both Wnt- and ErbB-dependent oncogenic signaling pathways. Among these underlying genetic changes are loss-of-function mutations in the adenomatous polyposis coli (APC) gene, which subsequently leads to the activation of beta-catenin and constitutive Wnt signaling. This is often followed by gain-of-function mutations in KRAS or BRAF proto-oncogenes. The RAS signaling pathway, operating through the RAF Ser/Thr kinase family, triggers the subsequent activation of mitogen-activated protein/extracellular signal-regulated kinase 1 and 2 (MEK1/2) and the extracellular signal-regulated kinase 1 and 2 (ERK1/2). This specific signaling cascade has garnered considerable attention due to the high frequency of KRAS and BRAF mutations detected in various human cancers. Activating mutations of KRAS, for example, are found in approximately 40% of advanced CRC cases. Furthermore, the BRAF valine 600 (BRAFV600E) mutation, which results in the constitutive activation of BRAF, is present in about 11% of CRCs and is associated with a poor prognosis for affected individuals. Given the challenges associated with the pharmacological inhibition of KRAS, alternative strategies that target downstream RAS effectors, such as RAF and MEK1, have been explored. However, these approaches have shown limited effectiveness as monotherapies for treating CRC, largely due to a feedback reactivation of MAPK signaling. This reactivation mechanism often involves the amplification of the driving oncogenes, KRAS or BRAF, in colorectal cells that have been treated with MEK1 inhibitors. Additional mechanisms encompass the EGFR/HER1-dependent reactivation of MAPK in BRAFV600E-mutated colorectal cancer cells exposed to a BRAF inhibitor. Similarly, MAPK reactivation in KRAS-mutated colorectal cancer cells subjected to MEK1 inhibition can also result from the induction of HER3. Clinical trials have been conducted in which RAF and EGFR, or RAF and MEK, are co-targeted in an attempt to suppress the feedback reactivation of MAPK signaling. While some patients initially experienced benefits from these combination therapies, resistance and disease recurrence invariably developed during disease progression. In these cases, resistant colorectal cancer cells exhibited KRAS or BRAF amplification, as well as the emergence of activating MEK1 mutations. The RAS-RAF-MEK1-ERK1/2 cascade critically relies on scaffold proteins, which serve to assemble pathway molecules, thereby regulating signaling. These scaffold proteins include Ras GTPase-activating-like protein (IQGAP1) and kinase suppressor of RAS (KSR). Another such protein is KIAA1199, now known as CEMIP (“Cell Migration-inducing and hyaluronan-binding protein”), which exhibits enhanced expression in cervical, breast, and colorectal cancer. CEMIP promotes cell survival and invasion, at least in part through EGFR-dependent MEK1 and ERK1/2 activation in cervical and breast cancer cells. However, it remains unclear which scaffold proteins, if any, are specifically implicated in MAPK reactivation in colorectal cells that exhibit intrinsic or acquired resistance to BRAF or MEK1 inhibitors. It is plausible that both Wnt- and MAPK-dependent signaling pathways are interconnected in the promotion of resistance to targeted therapies. This study identifies CEMIP as a MEK1-binding protein whose expression is induced by Wnt signaling. CEMIP promotes acquired resistance to MEK1 inhibition in BRAFV600E-mutated colorectal cancer cells, at least through ERK1/2 signaling and Myc. This CEMIP-dependent cascade is essential for amino acid synthesis in resistant cells. Collectively, these data define CEMIP as a key driver of resistance to MEK1 inhibition in BRAFV600E-mutated colorectal cancer, acting upstream of the ERK1/2 and Myc cascade.

Materials And Methods

Cell Culture And Reagents

Colorectal cancer cell lines, including HT-29, HCT116, SW480, and COLO-205, were obtained from the American Type Culture Collection (ATCC, Manassas, VA) in 2009. These cell lines were characterized by ATCC using a comprehensive database of short tandem repeat (STR) DNA profiles. Frozen aliquots of freshly cultured cells were prepared, and experiments were conducted using resuscitated cells that had been cultured for less than six months. All cell lines underwent mycoplasma testing to ensure the absence of contamination. HT-29 and HCT116 cells were cultured in McCoy’s 5A medium, supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; Gibco, Life Technologies, Naerum, Denmark) and 100 units/ml penicillin/streptomycin. SW480 cells were cultured in DMEM medium, supplemented with 10% HI-FBS, 1% glutamine, and 100 units/ml penicillin/streptomycin. COLO-205 cells were cultured in RPMI-1640 medium, supplemented with 10% HI-FBS, 1% glutamine, and 100 units/ml penicillin/streptomycin. Selumetinib (AZD6244), Vemurafenib (PLX4032, RG7204), PD98059, and PNU-74654 were obtained from Selleck Chemicals (Houston, TX, USA).

Intestinal Epithelial Cell Extraction And Ex-Vivo Organoid Cultures

Intestines and colons were extracted from C57BL/6 (Wnt OFF) or Apc+/Min (Wnt ON) mice. All studies conducted were approved by the Institutional Animal Care and Use Committee of the University of Liege. Bowels were washed for 10 minutes at 37°C in a PBS-DTT (1 mM) buffer and then incubated for 15 minutes at 37°C in a HBSS-EDTA buffer (30 mM). Cells were harvested, washed twice in PBS, and flash-frozen. For the generation of ex-vivo organoid cultures, small pieces of intestine were incubated in 2mM EDTA-PBS for 30 minutes at 4°C. Crypts were extracted, washed twice in PBS, and cultured in Matrigel (Biosciences, San Jose, CA, USA). DMEM/F12 medium, supplemented with EGF (20ng/ml), Noggin (100ng/ml), and R-Spondin (500ng/ml), was added every two days. Apc-mutated organoids were cultured in DMEM/F12 medium, supplemented with EGF (20ng/ml) and Noggin (100ng/ml), but without R-Spondin. The enrichment of Lgr5+ stem cells in ex-vivo organoids generated with intestinal crypts from C57BL/6 mice was achieved by treating them with a combination of Valproic acid (1 mM) and CHIR99021 (3 PM), a GSK3 inhibitor.

Generation Of Selumetinib-Resistant Colorectal Cancer Cell Lines (HT-29/SR, COLO-205/SR, SW480/SR And HCT116/SR) And Selumetinib-Resistant Ex-Vivo Organoids

Four colorectal cancer cell lines, namely HT-29, COLO-205, SW480, and HCT116, were used as parental cell lines (HT-29/P, COLO-205/P, SW480/P, and HCT116/P) from which the Selumetinib-resistant cell lines (HT-29/SR, COLO-205/SR, SW480/SR, and HCT116/SR) were generated. These resistant cell lines were established by repeatedly subculturing cells in the presence of incrementally increasing concentrations of Selumetinib, ranging from 0.05 to 1.5 μM for HT-29/P and SW480/P cells, from 0.05 to 2 μM for HCT116/P cells, and from 0.005 to 0.3 μM for COLO-205/P cells, over a period of six months. For the maintenance of Selumetinib-resistant colorectal cancer cell lines, the maximum concentration of Selumetinib, specifically 1.5 μM for HT-29/SR and SW480/SR cells, 2 μM for HCT116/SR cells, and 0.3 μM for COLO-205/SR cells, was added to the normal culture medium. For the generation of Selumetinib-resistant ex-vivo organoids, organoids generated from Apc+/Min mice were initially cultured with 1 PM of Selumetinib for two weeks. Subsequently, the concentration was increased by 0.5 PM every two weeks until reaching a final concentration of 5 PM.

Lentiviral Cell Infection

Control shRNA, CEMIP, Myc, TAK1, and FRA-1 shRNA lentiviral pLKO1-puro plasmid constructs were obtained from Sigma (St. Louis, MO, USA). Control shRNA and CEMIP shRNA lentiviral pLKO1-puro-IPTG-inducible plasmid constructs were also purchased from Sigma. Lentiviral infections were performed following established procedures.

For lentiviral infections of ex-vivo organoids, the organoids were manually disrupted and washed with PBS to remove any debris. Subsequently, they were trypsinized for 30 minutes at 37°C. After washing with PBS, the cells were washed using strainers (70 μM) with 20 ml of PBS and then centrifuged at 200 g for 5 minutes at 4°C. The resulting cell suspension was diluted in 500 microliters of full media optimized for organoid growth, and 500 microliters of infectious supernatants were added, mixed, and incubated in a CO2 incubator for 12 hours. Following incubation, the organoids were centrifuged for 5 minutes at 4°C, washed once with 1 ml of PBS, and plated as usual. Twenty-four hours later, full media containing 2 μg/ml of puromycin was added to select for transduced cells.

Quantitative Real-Time Pcr

Total RNAs were extracted using the E.Z.N.A Total RNA kit (Promega). Complementary DNAs (cDNAs) were synthesized using the Revert aid H minus reverse transcriptase kit (Thermo Scientific), and Real-time PCR analyses were performed as previously described. Messenger RNA (mRNA) levels in control organoids or cells were designated as 1, and mRNA levels in other experimental conditions were calculated relative to the control after normalization with E-Actin. The presented data are derived from at least two independent experiments, each performed in triplicate.

Mts Assay

Cells, counted using the TC20TM Automated Cell Counter (Bio-Rad, Pleasanton, CA, USA), were plated in 96-well flat-bottom plates at a density of 2000 cells per well in triplicate. They were then treated with varying concentrations of Selumetinib or Vemurafenib for a period of 72 hours. Cell viability was determined using the MTS assay reagent (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega, Madison, WI, USA) according to the manufacturer’s protocol. The absorbance was measured at 490 nm using a Wallac Victor2 1420 Multilabel counter (Perkin Elmer, Wellesley, MA). The absorbance of untreated cells was defined as 100%, and the number of viable cells in other experimental conditions was calculated relative to the untreated cells.

Clonogenic Assay

Cells were seeded in 60-cm dishes at a density of 3000 cells per dish in duplicate. Twenty-four hours after plating, various concentrations of Selumetinib or Vemurafenib were added to each dish. After treatment for 24 hours, the cells were washed with PBS and further incubated for 15 days to allow colony formation. Following incubation, the cells were stained with 0.5% crystal violet in 25% methanol-containing PBS. Colonies were examined under a light microscope and counted after capturing images.

Western Blot Analysis

Cells were lysed in a buffer containing 20 mM Tris-HCl, 0.5 M NaCl, Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycophosphate, 10 mM NaF, 300 μM Na3VO4, 1 mM Benzamidine, 2 μM PMSF, and 1 mM DTT. Western blots were performed as previously described, using antibodies listed.

Caspase-3/7 Activity Assay

Caspase-3/7 activity was quantified using the Caspase-3/7 Glo Assay (Promega). Cells were treated with Selumetinib or Vemurafenib for the indicated periods, and caspase-3/7 activity was quantified from cell lysates. Luminescence was measured at 490 nm using a Wallac Victor2 1420 Multilabel counter (Perkin Elmer). Luminescence values in vehicle-treated control samples were designated as 1, and values obtained in other experimental conditions were calculated relative to the vehicle control.

Extraction Of Cytoplasmic And Nuclear Proteins

Cells were incubated on ice for 10 minutes in cytoplasmic lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, NP-40 0.3% and Protease inhibitor). After centrifugation at 3000 rpm for 5 minutes at 4°C, the supernatant fraction (cytoplasmic extract) was harvested, and the pellet was resuspended in nuclear lysis buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, Glycerol 25%, and Protease inhibitor). This suspension was incubated on ice for 10 minutes and centrifuged at 14000 rpm for 5 minutes at 4°C, and the supernatant containing the nuclear fraction was retained.

Biochemical Fractionation

Cells were resuspended and homogenized in a Dounce homogenizer with the lysis buffer (150 mM NaCl, 5 mM DTT, 5 mM EDTA, 25 mM Tris HCl pH7.4, protease inhibitors) and centrifuged at 1000g for 10 minutes at 4°C. The supernatant was adjusted to 1% Triton X-100 and left on ice for 30 minutes. Four volumes of OptiPrep were added to two volumes of supernatant. OptiPrep was diluted with the lysis buffer plus 1% Triton X-100 to give 35, 30, 20, and 5% (w/v) iodixanol. 0.6 ml of each sample, as well as the four gradient solutions, were layered in tubes for the swinging-bucket rotor. Samples were centrifuged at 30000 rpm for 16 hours. Fractions of equal volume were collected for subsequent western blot analyses.

For the second fractionation experiment, the Optiprep Density Gradient centrifugation kit was purchased from Sigma Aldrich. Briefly, approximately 300 million HT-29 cells showing some acquired resistance to Selumetinib were trypsinized, washed in PBS, and centrifuged at 600 g for 5 minutes. Cells were then lysed with the extraction buffer, homogenized using the Dounce homogenizer, and centrifuged at 1000 g for 10 minutes. The supernatant was collected and centrifuged at 20000 g for 30 minutes. The pellet (which includes ER, lysosomes, peroxisomes, mitochondria, and endosomes) was diluted to a 19% Optiprep Density gradient solution and centrifuged on an OptiPrep Density Gradient at 100000 g for 8 hours. Fractions of equal volume were collected for subsequent western blot analyses.

Immunoprecipitation

Anti-MEK1, -BRAF, and -IgG (negative control) antibodies were first coupled non-covalently to a mixture of Protein A/G-Sepharose. The antibody-Protein A/G-Sepharose conjugates were then pelleted by centrifugation at 5,000 rpm for 2 minutes, the supernatant removed, and the beads washed with 0.1 M sodium borate pH 9.3. This was repeated four times, after which the beads were resuspended in 20 mM dimethyl pimelimidate dihydrochloride (DMP) freshly made in 0.1 M sodium borate pH 9.3 and gently mixed on a rotating wheel for 30 minutes at room temperature. Following centrifugation at 5,000 rpm for 2 minutes, supernatant was removed, and fresh 20 mM DMP/0.1 M sodium borate pH 9.3 solution was added to the beads, which were then gently mixed for a further 20 minutes. The beads were then spun down at 5,000 rpm for 2 minutes, the supernatant removed, and four washes with 50 mM glycine pH 2.5 carried out to remove any antibody coupled non-covalently. Afterwards, the beads were washed twice with 0.2 M Tris-HCl pH 8.0 (neutralization step) and then resuspended in the same solution and mixed gently on a rotating wheel at room temperature for 2 hours. The beads were then used immediately for immunoprecipitation analyses.

Kinase Assay

Control or CEMIP-depleted HT29 cells showing acquired resistance to Selumetinib were subjected to anti-MEK1 or -IgG (negative control) immunoprecipitation. Selumetinib was added as a control in some experimental conditions to inhibit MEK1 activity. The kinase assay was conducted at 30°C for 30 minutes with 1 μg of GST-ERK2 substrate (ThermoFisher Scientific), 10 μCi of [γ32P] ATP in 20 μl of kinase buffer (25 mM HEPES, pH 7.5, 10 mM MgCl2, 25 mM β-glycerophosphate, 1mM Na3VO4, and 1mM dithiothreitol). ERK2 phosphorylation was revealed by autoradiography.

Proximal Ligation And Chip Assays

Parental and resistant HT-29 cells, or control and CEMIP-depleted resistant HT-29 cells, were plated in 8-chamber cell culture dishes, and proximal ligation assays were performed following established procedures.

Chromatin immunoprecipitation (ChIP) was conducted using anti-TCF4 or IgG antibody as a negative control. A TCF4 binding site (site #1, located 839 bp upstream of the transcriptional start site on the CEMIP promoter) was identified through in silico analysis using MatInspector (Genomatix). TCF binding sites #2, #3, and #4, located 26417, 75344, and 79348 bp downstream of the transcriptional start site within intron 1, respectively, were previously described. A negative binding site was randomly selected in exon 1 of the CEMIP sequence. Primer sequences used are available upon request. Extracts from Selumetinib-resistant HT-29 cells, either left untreated or treated with 3PM Selumetinib for 24 hours, were precleared through an incubation step with protein A/BSA/Herring sperm DNA for 18 hours, and immunoprecipitations were performed overnight at 4°C with the relevant antibody, followed by a 1-hour incubation with protein A/BSA/Herring sperm DNA. Protein-DNA complexes were washed three times with high salt buffer (1% Triton X-100, 0.1% SDS, 500 mM NaCl, 2 mM EDTA pH 8.0, 20 mM Tris HCl pH 8.0, with protease inhibitors) and once with LiCl buffer (20 mM Tris HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 0.5 mM PMSF, and protease inhibitors). After elution, proteinase K treatment, and reversal of crosslinks, DNA fragments were analyzed by real-time PCR with SYBR Green detection. Values were calculated as ratios between ChIP signals obtained with the anti-TCF4 (specific) or IgG (non-specific) antibodies. Input DNA was analyzed simultaneously and used for normalization purposes.

Immunofluorescence

Immunofluorescence on cells was carried out following established procedures. For immunofluorescence on ex-vivo organoids, they were grown in 8-well chamber slides (Thermo Fisher Scientific, Lab-TekTM), fixed in 4% paraformaldehyde for 15 minutes, and washed twice in PBS. They were then incubated in permeabilization solution (PBS with 0.5% Triton X-100) for 15 minutes, washed in PBS, and incubated for 60 minutes in PBS containing 0.2% Triton X-100, 0.05% Tween, and 1% bovine serum albumin (BSA). Organoids were incubated overnight at 4°C with primary antibody in the same solution without Triton. After incubation, organoids were washed three times in PBS and incubated for 40 minutes with secondary antibody, washed three times with PBS, and incubated for 5 minutes with DAPI solution before being mounted with the ProLong Gold Antifade Mountant from Invitrogen. The anti-Ki-67 mouse monoclonal antibody was obtained from BD Biosciences (San Jose, CA, USA).

For immunofluorescence on cells expressing the SNAP-CEMIP construct, the CEMIP coding sequence was subcloned into the pSNAPf Vector (New England BioLabs, Ipswich, MA, USA), in which the SNAP tag is localized at the C-terminal end. HCT116 cells were transfected with controls (empty vector and pSNAPf -Cox8A) and CEMIP-SNAP plasmids and labeled with SNAP-Cell TMR-Star (New England BioLabs) 24 hours after transfection. Cells were fixed with 4% PAF and co-stained with endosomal or ER markers, as described previously. DAPI was used for nuclei staining.

Facs Analysis

Organoids were manually disrupted, washed with PBS to eliminate debris, and subsequently trypsinized for 30 minutes at 37°C. After washing with PBS, cells were washed via strainers (70 μM) with 20 ml of PBS and centrifuged (200 g) for 5 minutes at 4°C. They were then diluted in 500 microliters of PBS and incubated for 40 minutes on ice with anti-mouse CD24-PE eBioscience and anti-mouse CD133-APC eBioscience antibodies, washed once in PBS, and analyzed on the FACS CantoII. DAPI staining was used for selection of live cells.

Tumor Xenograft Experiments

HT-29/SR cells were infected with an IPTG-inducible CEMIP shRNA (HT-29/SR-iCEMIP shRNA) or control shRNA (HT-29/SR-iControl shRNA). HT-29/SR-iCEMIP shRNA or HT-29/SR-iControl shRNA cells (1.5 x 106), mixed with matrigel at a ratio of 1:1, were injected subcutaneously into the right or left flank of 6- to 8-week-old NOD/SCID male mice, respectively. Mice were monitored and tumors tracked via caliper measurements. Tumor volume was determined using the following formula: length x width x height x 0.5236 (n=4 mice/group). Mice were treated with a combination of 10 mM IPTG in their drinking water and 25 mM IPTG (200 μL) via intraperitoneal injection, and either left untreated or treated with Selumetinib (20 mg/kg) via intraperitoneal injection five days a week for two weeks. Mice from all treatment groups were euthanized, and tumors were excised and tissue archived for immunofluorescence and real-time PCR analysis.

Establishment Of Metabolomic Profiles By Targeted Metabolomics

For targeted metabolomics analysis of ex-vivo organoids, each sample was washed three times with cold PBS, collected into an Eppendorf tube, frozen in liquid nitrogen, and stored at -80°C until extraction. The extraction solution used consisted of 50% methanol, 30% ACN, and 20% water. The volume of extraction solution added was calculated based on the cell count (2 x 106 cells per ml). After the addition of the extraction solution, samples were vortexed for 5 minutes at 4°C and immediately centrifuged at 16,000 g for 15 minutes at 4°C. The supernatants were collected and analyzed by liquid chromatography–mass spectrometry using a SeQuant ZIC-pHilic column (Merck) for the liquid chromatography separation. Mobile phase A consisted of 20 mM ammonium carbonate plus 0.1% ammonia hydroxide in water. Mobile phase B consisted of ACN. The flow rate was maintained at 100 ml/minute, and the gradient was as follows: 0 minutes, 80% of B; 30 minutes, 20% of B; 31 minutes, 80% of B; and 45 minutes, 80% of B. The mass spectrometer (QExactive Orbitrap, Thermo Fisher Scientific) was operated in a polarity switching mode, and metabolites were identified using TraceFinder Software (Thermo Fisher Scientific). To ensure robust statistical analysis, metabolomics data were normalized using the median normalization method. The data were further pre-processed with a log transformation. MetaboAnalyst 3.0 software was used to conduct statistical analysis and heatmap generation, and an unpaired two-sample t-test was chosen to perform the comparisons. The algorithm for heatmap clustering was based on the Pearson distance measure for similarity and the Ward linkage method for biotype clustering. Metabolites with similar abundance patterns were positioned closer together.

Statistical Analysis

The two-tailed Student’s t-test was applied for statistical analysis when only two groups of interest were compared. Results were plotted as mean ± SD and were considered significant in all experiments at *** (p< 0.001), ** (p < 0.01), and * (p< 0.05).

Results

Ex-Vivo Organoids With Some Acquired Resistance To Mek1 Inhibition Show Enhanced Cemip Expression

Cancer stem cells are known to contribute to resistance to targeted therapies. To establish a link between cancer stem cells and acquired resistance to targeted therapies, ex-vivo organoids exhibiting constitutive Wnt signaling (i.e., generated with extracts of intestinal crypts from Apc+/Min mice) were subjected to increasing concentrations of Selumetinib to generate resistant organoids. The maintenance of these ex-vivo organoids relies on the self-renewal potential of cancer stem cells. Resistant organoids displayed a larger size but did not exhibit significant changes in cell proliferation, as determined by the percentage of Ki-67+ cells. They were also protected from caspase 3-dependent cell death, compared to parental organoids. The scaffold protein CEMIP may connect pro-tumorigenic Wnt- and MAPK signaling pathways, as it represents the most robust Wnt-induced gene candidate and is also required for MAPK activation upon activation of ErbB signaling. As such, CEMIP may actively contribute to acquired resistance to Selumetinib as a signaling protein involved in MAPK reactivation. Ex-vivo organoids treated with a combination of valproic acid and CHIR999021, a GSK3 inhibitor, to induce Wnt signaling, showed elevated mRNA levels of Wnt target genes, such as Lgr5 and CEMIP, while the level of Dclk1, a marker of differentiated Tuft cells, was downregulated. CEMIP induction by these drugs was also detected at the protein level. Immunofluorescence confirmed that treatment with valproic acid and CHIR999021, which enriches Lgr5+ cells in ex-vivo organoids, decreased the number of Dclk1+ Tuft cells. Therefore, CEMIP expression is transcriptionally induced by Wnt signaling. Importantly, resistant organoids showed increased CEMIP, SOX9, HER3, and BRAF expression, as well as enhanced activation of MEK1, ERK1/2, and mTOR, as determined by 4EBP1 phosphorylation. CEMIP was actually increased at the mRNA level in resistant organoids. Moreover, Myc, which controls protein synthesis and organ size, was increased at the protein but not mRNA levels. Of note, no mutations were detected on BRAF or MEK1 in these resistant organoids. Resistant organoids were enriched in CD24+/CD133+ cancer stem cells, which aligns with the upregulation of CD133 in colon cancer cells exhibiting hyperactivation of the RAS-RAF-MEK1 cascade. Therefore, Selumetinib-resistant organoids exhibit all molecular features classically associated with acquired resistance to MEK1 inhibition.

Cemip Is Connected To Erbb/Mek1-, Lef1- And Myc-Dependent Pathways In Colon Adenocarcinoma

To explore whether CEMIP links Wnt-dependent gene transcription to MEK1 signaling, CEMIP was depleted in BRAFV600E-mutated HT-29 colorectal cancer cells, and RNA-Seq experiments were carried out, combined with Gene Set enrichment analyses (GSEA). In agreement with previous observations, CEMIP expression was linked to ErbB/MEK1 signaling, as a signature of genes induced through ErbB2, KRAS, or MEK1 was lost upon CEMIP deficiency. Genes controlled by the transcription factor LEF1 were also identified to be regulated by CEMIP. An iRegulon analysis was then conducted to identify all genes co-expressed with CEMIP in colon adenocarcinoma, revealing 285 candidates. Interestingly, many of these candidates are regulated by the Myc family of transcription factors. An Ingenuity analysis was also performed on these 285 co-expressed genes, revealing a significant enrichment of genes controlled by p53, Myc, and E-catenin, among others. Therefore, CEMIP expression is linked to ErbB-, Myc-, and E-catenin-dependent pathways.

Cemip Promotes Myc Expression Through Erk1/2 Activation

To explore how CEMIP and Myc are linked, the consequences of CEMIP deficiency in ex-vivo organoids were assessed. Depletion of CEMIP in parental or Selumetinib-resistant organoids severely impaired their maintenance. Consistently, the pool of CD24+/CD133+ cancer stem cells was impaired upon CEMIP deficiency. CEMIP deficiency downregulated HER3, Cyclin D1, Cyclin D2, Myc, SOX9, and phosphorylated ERK1/2 levels. To explore whether the link between CEMIP and Myc was found in other experimental systems, BRAFV600E-mutated COLO205 cells with some acquired resistance to MEK1 inhibition were generated by subjecting parental cells to increasing concentrations of Selumetinib. Selumetinib-resistant COLO205 cells showed elevated CEMIP mRNA and protein levels, as well as increased levels of pMEK1/2, pERK1/2, and pRSK1. Importantly, CEMIP deficiency in these cells also impaired MEK1 and ERK1/2 activation and decreased protein levels of Myc. Of note, effects on MEK1 activation were largely due to decreased total levels of MEK1 in CEMIP-deficient cells, which was not the case in ex-vivo organoids. These Selumetinib-resistant COLO205 cells were also resistant to PD98059, another MEK1 inhibitor, as pERK1/2 levels barely decreased at high concentrations of this inhibitor. Here, CEMIP deficiency in these cells decreased pMEK1, pERK1/2, as well as Myc protein levels. Conversely, the ectopic expression of CEMIP alone in DLD-1 cells enhanced both pERK1/2 and Myc protein levels without impacting on Myc mRNA levels, and also protected from cell death triggered by Selumetinib. Therefore, CEMIP maintains Myc protein levels in multiple experimental models.

Cemip Expression Is Induced Through Braf, Erk1/2 And Fra-1 Upon Acquired Resistance In Brafv600e- But Not Krasg13d Or G12a-Mutated Colorectal Cancer Cells

CEMIP expression is increased in ex-vivo organoids as well as in BRAFV600E-mutated COLO205 cells, both exhibiting resistance to MEK1 inhibition. To investigate whether this phenomenon extends to other experimental models, parental HT-29 cells (HT-29/P) were cultured with increasing concentrations of Selumetinib, leading to the generation of highly resistant HT-29 cells (HT-29/SR). These cells exhibited decreased E-cadherin levels, suggesting that they underwent epithelial-mesenchymal transition (EMT), a known feature of chemoresistance. Importantly, CEMIP mRNA and protein levels were strongly induced in resistant HT-29 cells. The nuclear levels of transcription factors that drive CEMIP gene transcription, specifically NF-κB and AP-1 family members, were examined. Both p65 and FRA-1, but not BCL-3 and c-JUN levels, were increased in nuclear extracts from resistant HT-29 cells. Of note, cytoplasmic BRAF showed elevated levels in resistant cells, which reflects intrachromosomal amplification. These molecular changes persisted even under conditions in which cells were constantly cultured with Selumetinib. Therefore, CEMIP expression is induced in resistant HT-29 cells, in which mutated BRAFV600E and nuclear p65 and FRA-1 protein levels are increased. FRA-1, but not p65, was driving CEMIP expression in these cells, as FRA-1 deficiency, but not p65 deficiency, impaired CEMIP expression at both mRNA and protein levels. FRA-1 controls the expression of several candidates acting in the canonical Wnt pathway in colon cancer cells, such as WNT10, DKK-1, and DVL-1. Given that CEMIP transcription is robustly induced upon Wnt activation, it was hypothesized that FRA-1 indirectly controls CEMIP expression through Wnt signaling. FRA-1 deficiency indeed impaired nuclear E-catenin levels and both WNT10 and DKK-1, two E-catenin target genes downregulated upon FRA-1 deficiency in HT-29/SR cells. FRA-1 deficiency also triggered cell death of HT-29/SR cells, as judged by clonogenic assays, at least due to Caspase-3/7 activation. As CEMIP expression is induced in BRAFV600E-mutated cells, it was reasoned that a BRAF inhibitor may decrease CEMIP expression. CEMIP mRNA and protein levels were severely decreased in HT-29/SR cells subjected to Verumafenib. As a consequence, Vemurafenib triggered some cell death and interfered with the capacity of these cells to form colonies. Taken together, these results define BRAFV600E, FRA-1, and E-catenin as upstream actors that drive CEMIP transcription in BRAFV600E-mutated resistant colorectal cancer cells. Given that Selumetinib decreased CEMIP expression in both Selumetinib-resistant organoids and HT-29 cells, FRA-1 protein levels upon MEK1 inhibition in HT-29/SR cells were examined. FRA-1, but also p65, c-JUN, E-catenin, and TCF4, were downregulated upon MEK1 inhibition, while the epithelial marker E-cadherin was increased. Moreover, TCF4 promotes CEMIP expression, as CEMIP protein levels severely decreased upon TCF4 deficiency in Selumetinib-resistant HT-29 cells. Consistently, the treatment of two Selumetinib-resistant colon cancer cell lines with PNU-74654, which inhibits the Wnt/E-catenin pathway by blocking the interaction between E-catenin and TCF4, decreased CEMIP mRNA and protein levels. Both TCF4 and E-catenin were recruited to TCF binding sites located on the CEMIP promoter as well as on intron 1. Therefore, Selumetinib decreases CEMIP expression, at least by negatively regulating protein levels of both FRA-1 and TCF4.

To explore whether KRASG13D or G12A-mutated colorectal cancer-derived cell lines exhibiting some acquired resistance to Selumetinib also display elevated levels of CEMIP, parental HCT116 and SW480 cells were also treated with increasing concentrations of Selumetinib to generate resistant HCT116 and SW480 cells, respectively (HCT116/SR and SW480/SR). Both resistant HCT116 and SW480 cell lines did not upregulate CEMIP, in contrast to resistant HT-29 cells. Yet, ERK1/2 reactivation was observed in all resistant cells. RSK1 activity was also specifically induced in BRAFV600E- but not in KRASG13D or G12A-mutated resistant cells. Both p65 and FRA-1 were not dramatically induced in both HCT116 and SW480 resistant cells. Levels of another scaffold protein, IQGAP-1, remained unchanged in resistant HT-29 cells. BRAFV600E-mutated resistant HT-29 cells, and to a lesser extent KRASG13D-mutated HCT116 cells, also showed enhanced HER3 and MET reactivations. Finally, a somatic mutation in MEK1 exon 3 (H119R) was found in resistant HT-29 cells. The H119R mutation, among others in the same domain of MEK1, was demonstrated to underlie resistance to the MEK1 inhibitor, PD184352. Taken together, these data indicate that BRAFV600E- but not KRASG13D or G12A-mutated colorectal cancer cells reactivate MAPK signaling and potently induce CEMIP gene transcription upon acquired resistance to MEK1 inhibition.

Cemip Deficiency Sensitizes Brafv600e-Mutated Resistant Ht-29 Cells To Mek1 Inhibition

To explore whether CEMIP contributes to the resistance to Selumetinib in BRAFV600E-mutated colorectal cancer cells, CEMIP was depleted from HT-29/SR cells, and they were subjected to Selumetinib. MEK1 inhibition decreased CEMIP mRNA levels. CEMIP deficiency enhanced DNA damage, as evidenced by increased pH2A.X levels (S139), as well as Caspase-3/7-dependent apoptotic cell death upon Selumetinib treatment. CEMIP-depleted HT-29/SR cells generated fewer colonies when subjected to Selumetinib. To assess whether this was also relevant in vivo, xenograft experiments were conducted in immunodeficient mice with control and CEMIP-depleted HT-29/SR cells, and mice were subsequently treated with Selumetinib. CEMIP mRNA levels were expectedly decreased in cells infected with the inducible shRNA construct. Also, Selumetinib failed to significantly trigger tumor regression in vivo in mice injected with HT-29/SR cells. Importantly, CEMIP deficiency, combined with Selumetinib, caused significant tumor regression. This was due to DNA damage and apoptosis, as evidenced by anti-pH2A.X (S139) and cleaved caspase-3 immunofluorescence. CEMIP-deficient HT-29/SR cells showed lower levels of HER3, even after treatment with Selumetinib, and failed to maintain levels of phosphorylated ERK1/2 and RSK1 when treated with Selumetinib. Moreover, E-Cadherin levels increased upon CEMIP deficiency, indicating that CEMIP is required for EMT maintenance, a process linked to chemoresistance. CEMIP expression also decreased at the protein level in Selumetinib-treated cells, similar to FRA-1 and also to BRAF but not KRAS levels, suggesting again that BRAF and FRA-1 control CEMIP expression. Therefore, CEMIP contributes to the acquired resistance to Selumetinib, at least by promoting MEK1-ERK1/2 signaling.

Cemip Is An Endosomal Protein

Next, biochemical fractionation was performed to identify the cell compartments from which CEMIP contributes to MEK1 and ERK1/2 reactivation in resistant cells. CEMIP co-fractionated with EEA1 and APPL1, two markers of signaling endosomes, and to a lesser extent with lysosomal markers (Rab7 and LAMP2) and with Rab11, a recycling endosome marker. CEMIP also co-fractionated with PDI, an endoplasmic reticulum marker, as previously described. Phosphorylated forms of MEK1 were also detected in CEMIP-positive fractions, suggesting that signaling endosomes are critical for MEK1 reactivation. In contrast, CEMIP did not co-fractionate with Caveolin-1 and Flotillin-1, two lipid raft markers. To explore the specific endosomes in which CEMIP is mainly located, a second fractionation experiment was conducted in which organelles of interest (ER, peroxisomes, mitochondria, and endosomes) were enriched from cell extracts and separated on a gradient by ultracentrifugation. CEMIP primarily co-fractionated with EEA1+ endosomes and to a much lesser extent with Rab5/7+ or APPL1+ endosomes. A SNAP-CEMIP construct expressed in HCT116 cells also partially colocalized with EEA1+ endosomes and with the ER, as assessed by immunofluorescence. CEMIP was the only endosomal protein to be upregulated in HT-29/SR cells, as both EEA1 and APPL1 levels remained unchanged. CEMIP associated with MEK1 in HT-29/SR cells, as evidenced by co-immunoprecipitation, and BRAF more weakly bound MEK1 in resistant versus parental HT-29 cells, more likely due to disengagement. Of note, pERK1/2 levels were totally abolished after 0.5 and 1 hour of treatment with Selumetinib in both parental and resistant HT-29 cells, but pERK1/2 levels were again detectable after 24 hours of MEK1 inhibition. CEMIP actually contributes to MEK1 activity, as an anti-MEK1 immunoprecipitate from CEMIP-depleted HT-29/SR cells was less potent at phosphorylating ERK2. CEMIP failed to bind mutated BRAFV600E in resistant HT-29 cells. Although more BRAFV600E dimers were detected by the proximal ligation assay in resistant versus parental HT-29 cells, CEMIP was dispensable for BRAFV600E dimerization. Therefore, CEMIP is localized in several cell compartments and contributes to MEK1 reactivation from signaling endosomes in resistant BRAFV600E-mutated colorectal cancer cells as a MEK1-binding protein.

Cemip Promotes Metabolic Reprogramming Potentially Through Myc

To explore the biology downstream of CEMIP, the metabolomic signature of both parental and resistant organoids was established. Severe metabolic reprogramming was detected in resistant organoids, as they showed elevated levels of TCA intermediates (Fumarate, Malate, Citrate, and Succinate). Multiple nucleotides, whose synthesis relies on Myc, were increased in resistant organoids. Proline, whose degradation is inhibited by Myc, was detected at higher levels in resistant organoids. Moreover, levels of arachidonic acid, which is regulated by Myc in lung cancer, were also elevated in resistant organoids, as were levels of other unsaturated fatty acids, such as oleic acid, a candidate reported to be upregulated in colon cancer. Finally, levels of Cystathionine, which is generated by Cystathionine β-synthase (CBS), an enzyme downregulated in gastrointestinal and hepatocellular malignancies, were decreased in resistant organoids. Therefore, metabolic reprogramming is observed in Selumetinib-resistant intestinal organoids. Importantly, CEMIP expression contributes to this process, as the production of lactate as well as levels of multiple amino acids was impaired upon CEMIP deficiency in these ex-vivo resistant organoids. To better define CEMIP as an upstream regulator of Myc, it was reasoned that Myc deficiency would mimic CEMIP deficiency in Selumetinib-resistant ex-vivo organoids. Indeed, the depletion of Myc impaired ERK1/2 activation and also downregulated CEMIP protein but not mRNA levels, suggesting that CEMIP and Myc mutually post-transcriptionally control their expression. Myc was also critical for the maintenance of Selumetinib-resistant ex-vivo organoids, similar to CEMIP. Moreover, Myc depletion had a profound effect on the levels of multiple metabolites, as levels of lactate and numerous amino acids were significantly downregulated in Myc-depleted cells. A comparison of the metabolic signatures of Selumetinib-resistant ex-vivo organoids with depleted Myc or CEMIP confirmed that both proteins control the production of multiple metabolites, such as amino acids (Methionine, Threonine, Tryptophane, Valine, Proline, Histidine, Asparagine, Phenylalanine, IsoLeucine, Leucine, Glycine, and L-Alanine) and lactate, among other candidates. Therefore, CEMIP may promote the acquired resistance to MEK1 inhibition, in part by potentially regulating levels of specific metabolites via a Myc-associated signaling pathway.

Discussion

This study describes the characterization of CEMIP as an endosomal protein that links Wnt-dependent gene transcription to MEK1-ERK1/2 signaling to promote acquired resistance to MEK1 inhibition in BRAFV600E-mutated colorectal cancer cells. CEMIP expression is induced in resistant cells through BRAFV600E, MEK1, RSK1, and FRA-1, which provides a mechanism by which these signaling proteins promote resistance to inhibitors of RAS effectors. In addition, CEMIP regulates levels of multiple amino acids seen in resistant cells, at least through Myc.

Multiple transcription factors govern CEMIP transcription, including the NF-κB proteins BCL-3 and p65 in cervical cancer cells. Functional NF-κB and AP-1 binding sites were also identified on the CEMIP promoter in breast cancer cells. This study defines FRA-1, one member of the AP-1 family of transcription factors, as well as TCF4, as key drivers of CEMIP expression in resistant BRAFV600E-mutated colorectal cancer cells. The BRAF inhibitor, which indirectly turns off ERK1/2 activity, also decreases FRA-1 protein levels. This observation aligns with the fact that ERK1/2 signaling stabilizes FRA-1 by preventing its proteasome-dependent degradation in colorectal cancer cells. Therefore, interfering with ERK1/2 signaling downregulates CEMIP transcription, at least through the destabilization of FRA-1 in resistant BRAFV600E-mutated colorectal cancer cells. This signaling cascade critically drives CEMIP transcription to establish a positive loop, as CEMIP physically binds MEK1 (but not BRAF) to sustain MEK1 activity.

The resistant BRAFV600E-mutated colorectal cancer cells in this study have several features linked to acquired resistance. They exhibit enhanced phosphorylation of MET and HER3, elevated levels of BRAFV600E, as well as a MEK1 mutation, all events contributing to ERK1/2 reactivation. The upregulation of CEMIP has been detected in all tested BRAFV600E- but not KRASG13D or G12A-mutated colorectal cancer cells exhibiting some acquired resistance to MEK1 inhibition. Yet, the induction of CEMIP upon acquired resistance was more severe in resistant BRAFV600E-mutated colorectal cancer HT-29 cells, in which the MEK1 mutation within exon 3 (H119R) was found. Therefore, the combination of both BRAFV600E and MEK1 mutations may be key genetic events to efficiently drive CEMIP expression upon acquired resistance.

CEMIP is found in signaling endosomes and is essential for ERK1/2 reactivation in BRAFV600E- but not KRASG13D or G12A-mutated cells, at least through binding to MEK1. Whether or not ERK1/2 activation downstream of tyrosine kinase receptors occurs from endosomes or from the cytoplasmic membrane has been the subject of an intense debate. While some studies support the notion that MAPK scaffold complexes found in endosomes are critical for signal transduction, other reports state that signaling from a tyrosine kinase receptor occurs from the cytoplasmic membrane. In support of this later hypothesis, EGFR endocytosis in endosomes helps to terminate Ras-dependent signaling to ERK1/2, as endogenous Ras is primarily located at the cytoplasmic membrane in low EGFR-expressing cells. BRAFV600E does not bind CEMIP, which aligns with the hypothesis that CEMIP only binds signaling proteins, such as EGFR or MEK1, found in endosomes, but not candidates, such as BRAF, which is activated at the cytoplasmic membrane. The enhanced CEMIP expression that is specifically observed in resistant BRAFV600E-mutated colorectal cancer cells may help to recycle HER3 and MET at the cytoplasmic membrane to sustain ERK1/2 signaling and/or to favor the assembly of a specific endosomal signaling platform for ERK1/2 reactivation.

A previous study demonstrated that a pool of CEMIP can be found in the ER. These results, combined with the present study revealing CEMIP in endosomes, raise questions on the mechanisms by which a scaffold protein localized in two distinct cell compartments promotes survival and chemoresistance. The answer may lie in the existence of membrane contacts between endosomes and the ER, a process that contributes to EGFR-dependent signaling. These physical contacts may help CEMIP to bring signaling proteins together to sustain ERK1/2 activation in resistant BRAFV600E-mutated colorectal cancer cells.

One key mechanism through which CEMIP deficiency circumvents the acquired resistance to MEK1 inhibition in BRAFV600E-mutated colorectal cancer cells may be through Myc, which is in agreement with the fact that the pharmacological inhibition of Myc circumvents the acquired resistance to c-Met inhibition. The correlative metabolic data show that Myc is a key effector downstream of CEMIP, 10074-G5 as CEMIP and Myc similarly control the production of multiple metabolites, including lactate, as well as amino acids, such as Glycine, which has been defined as a driver of cancer pathogenesis. It has been demonstrated that Selumetinib-resistant ex-vivo organoids exhibit high levels of multiple amino acids, which can be metabolized as a source of carbon and nitrogen for biosynthesis of fatty acids, lipids, nucleotides, and proteins to support proliferation and survival. Essential amino acids, such as Leucine, Tryptophane, and Phenylalanine, whose levels are controlled by both CEMIP and Myc in Selumetinib-resistant organoids, have been defined as signaling molecules for mTOR activation. Therefore, CEMIP and its downstream effector Myc may indirectly control mTOR signaling through the production of specific essential amino acids to support acquired resistance to MEK1 inhibition.

In conclusion, this study defines the scaffold and endosomal protein CEMIP as an upstream regulator of Myc that links Wnt- and MEK1-dependent signaling pathways. As CEMIP is linked to Myc and to specific metabolic reprogramming observed in resistant cells, this oncogenic pathway may hold therapeutic interest.