KRAS Mutant Colorectal Cancer Cell Lines Research Articles

A machine learning and drug repurposing approach to target ferroptosis in colorectal cancer stratified by sex and KRAS.

The landscape of sex differences in Colorectal Cancer (CRC) has not been well characterized with respect to the mechanisms of action for oncogenes such as KRAS. However, our recent study showed that tumors from male patients with KRAS mutations have decreased iron-dependent cell death called ferroptosis. Building on these findings, we further examined ferroptosis in CRC, considering both sex of the patient and KRAS mutations, using public databases and our in-house CRC tumor cohort. Through subsampling inference and variable importance analysis (VIMP), we identified significant differences in gene expression between KRAS mutant and wild type tumors from male patients. These genes suppress (e.g., SLC7A11 ) or drive (e.g., SLC1A5 ) ferroptosis, and these findings were further validated with Gaussian mixed models. Furthermore, we explored the prognostic value of ferroptosis regulating genes and discovered sex- and KRAS-specific differences at both the transcriptional and metabolic levels by random survival forest with backward elimination algorithm (RSF-BE). Of note, genes and metabolites involved in arginine synthesis and glutathione metabolism were uniquely associated with prognosis in tumors from males with KRAS mutations. Additionally, drug repurposing is becoming popular due to the high costs, attrition rates, and slow pace of new drug development, offering a way to treat common and rare diseases more efficiently. Furthermore, increasing evidence has shown that ferroptosis inhibition or induction can improve drug sensitivity or overcome chemotherapy drug resistance. Therefore, we investigated the correlation between gene expression, metabolite levels, and drug sensitivity across all CRC primary tumor cell lines using data from the Genomics of Drug Sensitivity in Cancer (GDSC) resource. We observed that ferroptosis suppressor genes such as DHODH , GCH1 , and AIFM2 in KRAS mutant CRC cell lines were resistant to cisplatin and paclitaxel, underscoring why these drugs are not effective for these patients. The comprehensive map generated here provides valuable biological insights for future investigations, and the findings are supported by rigorous analysis of large-scale publicly available data and our in-house cohort. The study also emphasizes the potential application of VIMP, Gaussian mixed models, and RSF-BE models in the multi-omics research community. In conclusion, this comprehensive approach opens doors for leveraging precision molecular feature analysis and drug repurposing possibilities in KRAS mutant CRC.

KRAS-mutant colorectal cancer cell lines cause a prothrombotic state through the upregulation of thrombin: experimental study

Background: The KRAS genotype status is strongly associated with a prothrombotic state in colorectal cancer, and hypercoagulability and cancer-related thrombosis are among the significant events leading to poor prognosis. However, this correlation has not been confirmed at the cellular level. This study aimed to assess the maximum platelet aggregation rate and thrombin expression induced by colorectal cancer cells under different KRAS genotypes. Materials and methods: Platelet aggregation rate assay and western blotting analysis were used to detect platelet aggregation and thrombin expression induced by four colorectal cancer cells with different KRAS genotypes, including RKO, HCT116, SW480, and SW620. FVIIa/tissue factor and thrombin inhibitors were added to explore changes in platelet aggregation rates induced by colorectal cancer cells and the association between KRAS genotype status and hypercoagulable state. Results: KRAS-mutant cells were more likely to increase maximal platelet aggregation, with RKO, HCT116, SW480, and SW620 inducing 34.7%, 55.4%, 44.4%, and 63.8% of platelet aggregation, respectively. The maximum platelet aggregation rate was higher in the metastatic rectal cancer tumour strain SW620 than in the primary rectal cancer strain SW480. RKO cells had lower thrombin expression than the other three cells. Furthermore, the addition of thrombin inhibitors caused a more significant decrease in the platelet aggregation rate in KRAS-mutant cell lines compared to KRAS wild-type cell lines. Conclusion: Compared to KRAS wild-type colorectal cancer cells, KRAS-mutant colorectal cancer cell lines were more likely to be hypercoagulable through the upregulation of thrombin expression, which was mainly achieved through the TF-thrombin pathway.

Osthole inhibits malignant phenotypes and induces ferroptosis in KRAS-mutant colorectal cancer cells via suppressing AMPK/Akt signaling.

Ferroptosis is a form of cell death driven by iron-dependent lipid peroxidation. Intriguingly, KRAS-mutant cancers are particularly vulnerable to ferroptosis. Osthole is a natural coumarin extracted from Cnidium spp. and other Apiaceous plants. In the present study, we explored the antitumor potential of osthole in KRAS-mutant colorectal cancer (CRC) cells. Cell viability assay, EdU incorporation assay, flow cytometry, tumor xenograft model, western blot, immunochemistry staining, immunofluorescence, transcriptome RNA sequencing and quantitative reverse transcription-PCR were performed to evaluate the influence of osthole treatment on KRAS-mutant CRC cells. We found that osthole treatment suppressed proliferation and tumor growth of KRAS-mutant CRC cell lines HCT116 and SW480. Moreover, osthole treatment increased ROS production and induced ferroptosis. Osthole treatment also promoted autophagy, but inhibition of autophagy by ATG7 knockdown or 3-MA showed no influence on osthole-induced ferroptosis. In comparison, osthole increased lysosomal activation, and co-treatment with lysosome inhibitor Baf-A1 attenuated osthole-induced ferroptosis. Besides, osthole treatment reduced the phosphorylation of AMPK, Akt and mTOR in HCT116 and SW480 cells, while restored AMPK signaling by AMPK agonist AICAR partially abrogated ferroptosis induced by osthole treatment. Finally, co-treatment with osthole increased the cytotoxicity of cetuximab in KRAS-mutant CRC cells in vitro and in vivo. Our results suggested that the natural product osthole exerted its anticancer effects in KRAS-mutant CRC cells via inducing ferroptosis, and this was partially through inhibiting AMPK/Akt/mTOR signaling. Our results may expand our current knowledge for the use of osthole as an anticancer agent.

Abstract 3187: Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome

Abstract Background: KRAS is commonly mutated in solid tumors. Cancer-associated fibroblasts (CAFs) are a key tumor stroma component, which can affect cancer progression and drug response. We hypothesized that defining the differential secreted proteins between CAFs and KRAS mutant-cancer cells could reveal potential mechanisms by which CAFs influence cancer progression and drug resistance. Methods: We studied 12 cell lines: 3 KRAS mutant-colorectal cancer cell lines (H747, LIM2099 and SW620), 3 KRAS mutant-lung cancer cell lines (H1792, H2030 and H23), 3 KRAS mutant-pancreatic cancer cell lines (CAPAN1, DANG, MIAPACA2) and 3 CAF cell line models (colorectal and lung CAF and pancreatic stellate cells). We quantified proteins in cell lines and serum-free media after 24 hour incubation in cells using mass spectrometry in a multiplexed manner (12plex) in triplicates to characterize the proteome and the profile of secreted proteins (secretome), respectively. Prediction software (signalP, secretomeP, TMHMM) and databases (surfaceome and cell surface protein atlas) were used to annotate secreted or transmembrane proteins. Results: Our proteome analysis quantified 9307 proteins. T-test between the cell types (CAF vs cancer) identified 1770 differentially expressed proteins, where 712 proteins were CAF-enriched. This included known CAF markers (e.g α smooth muscle actin) but also proteins not known to be CAF markers such as heat shock protein beta-5/6 (HSPB5/6). The secretome analysis identified 2330 secreted or transmembrane proteins where 202 were differentially expressed between cancer cells and CAFs. 190 proteins were CAF-enriched with the highest ranking being syndecan-2 (SDC2), collagen α-28(I) chain (COL28A1), decorin (DCN) and wnt family member 5B (WNT5B). Interestingly, basal proteome analysis of KRAS mutant-cancer cells showed expression of receptors corresponding to the CAF-enriched ligands, such as receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2) which respond to WNT5B. This suggests that CAF-enriched ligands may activate certain downstream pathways, including non-canonical WNT signaling, in KRAS mutant-cancer cells. Conclusion: We undertook deep quantitative proteomics where we identified potential novel CAF markers HSPB5/6. We have also found proteins in the CAF secretome, such as WNT5B, which may drive drug resistance in KRAS mutant-cancer cells. Citation Format: Rachel Lau, Lu Yu, Theodoros Roumeliotis, Lisa Pickard, Adam Stewart, Jyoti Choudhary, Udai Banerji. Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3187.

Cetuximab promotes RSL3-induced ferroptosis by suppressing the Nrf2/HO-1 signalling pathway in KRAS mutant colorectal cancer

Cetuximab is approved for the treatment of metastatic colorectal cancer (mCRC) with RAS wild-type. Nevertheless, the prognosis remains poor and the effectiveness of cetuximab is limited in KRAS mutant mCRC. Recently, emerging evidence has shown that ferroptosis, a newly discovered form of nonapoptotic cell death, is closely related to KRAS mutant cells. Here, we further investigated whether cetuximab-mediated regulation of p38/Nrf2/HO-1 promotes RSL3-induced ferroptosis and plays a pivotal role in overcoming drug resistance in KRAS mutant colorectal cancer (CRC). In our research, we used two KRAS mutant CRC cell lines, HCT116 and DLD-1, as models of intrinsic resistance to cetuximab. The viability of cells treated with the combination of RSL3 and cetuximab was assessed by the CCK-8 and colony formation assays. The effective of cetuximab to promote RSL3-induced ferroptosis was investigated by evaluating lipid reactive oxygen species accumulation and the expression of the malondialdehyde and the intracellular iron assay. Cetuximab therapy contributed to regulating the p38/Nrf2/HO-1 axis, as determined by western blotting and transfection with small interfering RNAs. Cetuximab promoted RSL3-induced ferroptosis by inhibiting the Nrf2/HO-1 in KRAS mutant CRC cells, and this was further demonstrated in a xenograft nude mouse model. Our work reveals that cetuximab enhances the cytotoxic effect of RSL3 on KRAS mutant CRC cells and that cetuximab enhances RSL3-induced ferroptosis by inhibiting the Nrf2/HO-1 axis through the activation of p38 MAPK.

Targeting antioxidant enzymes enhances the therapeutic efficacy of the BCL-XL inhibitor ABT-263 in KRAS-mutant colorectal cancers

Cancer chemotherapeutic drugs exert cytotoxic effects by modulating intracellular reactive oxygen species (ROS) levels. However, whether ROS modulates the efficacy of targeted therapeutics remains poorly understood. Previously, we reported that upregulation of the anti-apoptotic protein, BCL-XL, by KRAS activating mutations was a potential target for KRAS-mutant colorectal cancer (CRC) treatment. Here, we demonstrated that the BCL-XL targeting agent, ABT-263, increased intracellular ROS levels and targeting antioxidant pathways augmented the therapeutic efficacy of this BH3 mimetic. ABT-263 induced expression of genes associated with ROS response and increased intracellular ROS levels by enhancing mitochondrial superoxide generation. The superoxide dismutase inhibitor, 2-methoxyestradiol (2-ME), exhibited synergism with ABT-263 in KRAS-mutant CRC cell lines. This synergistic effect was attributed to the inhibition of mTOR-dependent translation of the anti-apoptotic MCL-1 protein via caspase 3-mediated cleavage of AKT and S6K. In addition, combination treatment of ABT-263 and 2-ME demonstrated a synergistic effect in in vivo patient-derived xenografts harboring KRAS mutations. Our data suggest a novel role for ROS in BH3 mimetic-based targeted therapy and provide a novel strategy for treatment of CRC patients with KRAS mutations.

In-Vitro and In-Silico Evaluations of Heterocyclic-Containing Diarylpentanoids as Bcl-2 Inhibitors Against LoVo Colorectal Cancer Cells.

In the present study, we investigated the in-vitro anti-cancer potential of six diarylpentanoids against a panel of BRAF- and KRAS-mutated colorectal cancer cell lines including T84, SW620, LoVo, HT29, NCI-H508, RKO, and LS411N cells. Structure–activity relationship study suggested that the insertions of tetrahydro-4H-thiopyran-4-one and brominated phenyl moieties are essential for better cytotoxicity. Among the evaluated analogs, 2e has been identified as the lead compound due to its low IC50 values of approximately 1 µM across all cancer cell lines and high chemotherapeutic index of 7.1. Anti-proliferative studies on LoVo cells showed that 2e could inhibit cell proliferation and colony formations by inducing G2/M cell cycle arrest. Subsequent cell apoptosis assay confirmed that 2e is a Bcl-2 inhibitor that could induce intrinsic cell apoptosis by creating a cellular redox imbalance through its direct inhibition on the Bcl-2 protein. Further molecular docking studies revealed that the bromophenyl moieties of 2e could interact with the Bcl-2 surface pocket through hydrophobic interaction, while the tetrahydro-4H-thiopyran-4-one fragment could form additional Pi-sulfur and Pi-alkyl interactions in the same binding site. In all, the present results suggest that 2e could be a potent lead that deserves further modification and investigation in the development of a new Bcl-2 inhibitor.

Synthesis and in-vitro anti-cancer evaluations of multi-methoxylated asymmetrical diarylpentanoids as intrinsic apoptosis inducer against colorectal cancer

In the present study, a series of nine stable 3,4,5-methoxylphenyl-containing asymmetrical diarylpentanoids, derivatives of curcuminoids, have been synthesized, characterized and evaluated for their in-vitro anti-cancer potential against a panel of BRAF- and KRAS-mutated colorectal cancer cell lines including T84, LoVo and SW620, HT29, RKO and NCI-H508, respectively. Structure-activity relationship study on cytotoxicity of tested compounds suggested that the presence of meta-hydroxyl and adjacent dimethoxyl groups are crucial for enhanced cytotoxicity of diarylpentanoids. Among the evaluated analogs, 8 has been identified as the lead compound due to its highest chemotherapeutic index of 9.9 and nano molar scale cytotoxicity against SW620 and RKO. Colonies formation and cell cycle analyses on 8-treated RKO cells showed that 8 exhibits strong anti-proliferative activity by inducing G2/M-phase cell arrest. Subsequent flow cytometry based annexin-V and DCFHDA studies suggested that 8 could induce apoptosis through intracellular ROS-dependent pathway. Further Western blot studies confirmed that 8 has induced intrinsic apoptosis in RKO cells through the up-regulations of Bad and Bax pro-apoptotic proteins and down-regulations of Bcl-2 and Bcl-xL pro-survival proteins. In all, the present results suggest that 8 could be a potent lead which deserves further modification and investigation in the development of small molecule-based anti-colorectal cancer agents.

Abstract LB-287: Combination therapies with CDK4/6 inhibitors to treat KRAS-mutant pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) patients have a dismal five-year survival rate of just eight percent in the advanced metastatic setting. Outcomes with standard chemotherapy regimens are less than ideal; therefore, the development of targeted therapies for the treatment of PDAC is a significant unmet clinical need. The two most frequent genetic events in PDAC (mutational activation of KRAS and loss of the tumor suppressor CDKN2A) converge on activation of the kinases CDK4 and CDK6, which promote G1 cell cycle progression. While clinically relevant agents directly targeting KRAS in PDAC remain elusive, pharmacologic restoration of CDKN2A function by inhibition of CDK4/6 may be an effective anti-KRAS therapeutic strategy. Supporting this hypothesis, we found that CDK4/6 inhibitors (e.g., palbociclib, abemaciclib, lerociclib) elicited single-agent activity in a subset of tested PDAC cell lines. However, using Reverse Phase Protein Array (RPPA), we observed widespread compensatory changes that overcome CDK4/6 inhibition, including increased anti-apoptotic protein expression, PI3K-mTOR and ERK MAPK signaling. Concurrent treatment with the potent and selective ERK1/2 inhibitor (ERKi) SCH772984 reversed resistance and increased sensitivity to palbociclib. This combination caused synergistic reduction in anchorage-dependent cell growth, and increased apoptosis, G1 arrest, and senescence. In an organoid model of pancreatic cancer, cell viability was synergistically reduced and caspase activation enhanced. We have also evaluated the combination of ERKi with palbociclib or the clinical-stage CDK4/6 inhibitor lerociclib (G1T38) in mouse models of PDAC, supporting our initiation of a phase Ib clinical trial of the ERK inhibitor ulixertinib/BVD-523 in combination with palbociclib in advanced metastatic pancreatic cancer (NCT03454035). Next, we sought to identify additional genes that regulate sensitivity to CDK4/6 inhibition. Using a CRISPR/Cas9 loss-of-function screen, we individually silenced expression of 2500 genes from the “druggable genome” in combination with palbociclib in six KRAS-mutant pancreatic and two KRAS-mutant colorectal cancer cell lines. We identified a wide array of genes that enhanced growth suppression in combination, centered around diverse signaling nodes including PI3K-AKT-mTOR signaling, cell cycle regulation and mitosis, SRC family kinase signaling, cell metabolism and biosynthesis, chromosome regulation and maintenance, and DNA damage & repair pathways, suggesting novel small molecule combinations to overcome de novo or acquired CDK4/6 inhibitor resistance in the clinic. We also identified genes whose loss imparts a survival advantage, suggesting possible resistance mechanisms to single-agent CDK4/6 inhibition. In total, these data suggest that CDK4/6 inhibitors alone, or in combination, may benefit PDAC patients clinically. Citation Format: Craig M. Goodwin, Sehrish Javaid, Andrew M. Waters, Bjoern Papke, Runying Yang, Mariaelena Pierobon, Daniel M. Freed, Patrick J. Roberts, Adrienne D. Cox, Kris C. Wood, Emanuel F. Petricoin, Autumn J. McRee, Channing J. Der. Combination therapies with CDK4/6 inhibitors to treat KRAS-mutant pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-287.

Receptor tyrosine kinase-dependent PI3K activation is an escape mechanism to vertical suppression of the EGFR/RAS/MAPK pathway in KRAS-mutated human colorectal cancer cell lines

BackgroundPrevious studies showed that the combination of an anti-Epidermal growth factor (EGFR) and a MEK-inhibitor is able to prevent the onset of resistance to anti-EGFR monoclonal antibodies in KRAS-wild type colorectal cancer (CRC), while the same combination reverts anti-EGFR primary resistance in KRAS mutated CRC cell lines. However, rapid onset of resistance is a limit to combination therapies in KRAS mutated CRC.MethodsWe generated four different KRAS mutated CRC cell lines resistant to a combination of cetuximab (an anti-EGFR antibody) and refametinib (a selective MEK-inhibitor) after continuous exposure to increasing concentration of the drugs. We characterized these resistant cell lines by evaluating the expression and activation status of a panel of receptor tyrosine kinases (RTKs) and intracellular transducers by immunoblot and qRT-PCR. Oncomine comprehensive assay and microarray analysis were carried out to investigate new acquired mutations or transcriptomic adaptation, respectively, in the resistant cell lines. Immunofluorescence assay was used to show the localization of RTKs in resistant and parental clones.ResultsWe found that PI3K-AKT pathway activation acts as an escape mechanism in cell lines with acquired resistance to combined inhibition of EGFR and MEK. AKT pathway activation is coupled to the activation of multiple RTKs such as HER2, HER3 and IGF1R, though its pharmacological inhibition is not sufficient to revert the resistant phenotype. PI3K pathway activation is mediated by autocrine loops and by heterodimerization of multiple receptors.ConclusionsPI3K activation plays a central role in the acquired resistance to the combination of anti-EGFR and MEK-inhibitor in KRAS mutated colorectal cancer cell lines. PI3K activation is cooperatively achieved through the activation of multiple RTKs such as HER2, HER3 and IGF1R.

2O - Receptor tyrosine kinase dependent PI3K activation is an escape mechanism to vertical suppression of the EGFR/RAS/MAPK pathway in KRAS-mutated colorectal cancer cell lines

2O - Receptor tyrosine kinase dependent PI3K activation is an escape mechanism to vertical suppression of the EGFR/RAS/MAPK pathway in KRAS-mutated colorectal cancer cell lines

PO-280 Mutant KRAS mediates fibroblast-induced colorectal cancer cell invasion

IntroductionKRAS is the most frequently mutated oncogene in colorectal cancer (CRC), being a potent initiator of tumorigenesis, a strong inductor of malignancy, and a predictive biomarker of non-response to anti-EGFR therapies. As such, extensive research has been done to exploit KRAS and its downstream signalling effectors as therapeutic targets. However, KRAS proved difficult to target, and inhibition of its signalling effectors has never resulted in significant clinical responses, highlighting the need for a better understanding of KRAS-associated signals. Since the tumour microenvironment plays a key role in tumour aggressiveness, research on this area became an attractive alternative as new targets for therapy may arise from the study of cancer cell-microenvironment crosstalk. The aim of this study was to characterise, at the molecular and functional levels, the role of mutant KRAS in mediating CRC cells-fibroblasts crosstalk.Material and methodsUsing fibroblasts-conditioned media (CM) as a chemoattractant, we performed matrigel invasion assays with KRAS mutant CRC cell lines in which we silenced KRAS using siRNA. Additionally, we performed ELISA assays to quantify the levels of fibroblasts-secreted factors and resorting to western blot we evaluated the expression of some cell surface proteins.Results and discussionsBy performing in vitro invasion assays we observed that the CM promoted CRC cell invasion in a KRAS-dependent manner. Analysis of the CM for the detection of pro-invasive factors, revealed the presence of high levels of HGF. Accordingly, neutralisation of HGF in the fibroblasts CM abrogated CRC invasion, and supplementation of control CM with HGF induced invasion in a KRAS-dependent manner. Additionally, we have also observed that KRAS regulates the expression of HGF receptor, C-MET, along with other C-MET co-receptors.ConclusionIn conclusion, our results show that KRAS may be an important modulator of response to fibroblasts-secreted factors that induce CRC cells invasion. Therefore, this work suggests that targeting of C-MET can be a useful tool to abrogate invasion of KRAS mutant tumours and sets a rational to test C-MET inhibitors in the treatment of KRAS mutant CRC patients, who currently lack effective therapeutic options.

A miR-29b Byproduct Sequence Exhibits Potent Tumor-Suppressive Activities via Inhibition of NF-κB Signaling in KRAS-Mutant Colon Cancer Cells.

We previously demonstrated that miR-29b-3p is a hopeful miRNA-based therapy against colorectal cancer. In this study, we aimed to clarify a value of miR-29b-1-5p as a next-generation treatment, especially for KRAS-mutant colorectal cancer. RT-PCR assay showed that the expression of miR-29b-3p was high, and its partner strand, miR-29b-1-5p, level was only negligible in clinical colorectal cancer samples. Mimic-miR-29b-1-5p significantly inhibited proliferation of KRAS-mutant colorectal cancer cell lines DLD1 and SW480 and KRAS wild-type HT29 cells. Proliferative activity was further examined by either miR-29b-1-5p strand or its opposite complementary sequence because miR-29b-1-5p is a passenger miRNA and may have no physiologic function. We found that completely opposite complementary strand to miR-29b-1-5p, but not miR-29b-1-5p, possessed a potent antitumor effect and named this byproduct miRNA sequence "MIRTX." MIRTX directly targeted the 3'-UTR of CXCR2 and PIK3R1 mRNA and suppressed the NF-κB signaling pathway in KRAS-mutated colorectal cancer cells. MIRTX induced apoptosis in DLD1 with downregulation of antiapoptotic BCL2, BCL-xL, and MCL1 and upregulation of cleaved caspase-3 and cleaved PARP. In mouse xenograft models, systemic administration of MIRTX using a super carbonate apatite as a delivery vehicle significantly inhibited tumor growth of DLD1 and HT29 cells without any particular toxicities. In conclusion, these findings indicate that inhibition of NF-κB signaling by this novel miRNA-based therapeutic could be a promising treatment against refractory KRAS-mutant colorectal cancer and KRAS wild-type colorectal cancer. Mol Cancer Ther; 17(5); 977-87. ©2018 AACR.

Comprehensive Analysis of miRNome Alterations in Response to Sorafenib Treatment in Colorectal Cancer Cells.

MicroRNAs (miRNAs) are master regulators of drug resistance and have been previously proposed as potential biomarkers for the prediction of therapeutic response in colorectal cancer (CRC). Sorafenib, a multi-kinase inhibitor which has been approved for the treatment of liver, renal and thyroid cancer, is currently being studied as a monotherapy in selected molecular subtypes or in combination with other drugs in metastatic CRC. In this study, we explored sorafenib-induced cellular effects in Kirsten rat sarcoma viral oncogene homolog olog (KRAS) wild-type and KRAS-mutated CRC cell lines (Caco-2 and HRT-18), and finally profiled expression changes of specific miRNAs within the miRNome (>1000 human miRNAs) after exposure to sorafenib. Overall, sorafenib induced a time- and dose-dependent growth-inhibitory effect through S-phase cell cycle arrest in KRAS wild-type and KRAS-mutated CRC cells. In HRT-18 cells, two human miRNAs (hsa-miR-597 and hsa-miR-720) and two small RNAs (SNORD 13 and hsa-miR-3182) were identified as specifically sorafenib-induced. In Caco-2 cells, nine human miRNAs (hsa-miR-3142, hsa-miR-20a, hsa-miR-4301, hsa-miR-1290, hsa-miR-4286, hsa-miR-3182, hsa-miR-3142, hsa-miR-1246 and hsa-miR-720) were identified to be differentially regulated post sorafenib treatment. In conclusion, we confirmed sorafenib as a potential anti-neoplastic treatment strategy for CRC cells by demonstrating a growth-inhibitory and cell cycle–arresting effect of this drug. Changes in the miRNome indicate that some specific miRNAs might be relevant as indicators for sorafenib response, drug resistance and potential targets for combinatorial miRNA-based drug strategies.

SLC25A22 Promotes Proliferation and Survival of Colorectal Cancer Cells With KRAS Mutations and Xenograft Tumor Progression in Mice via Intracellular Synthesis of Aspartate

Many colorectal cancer (CRC) cells contain mutations in KRAS. Analyses of CRC cells with mutations in APC or CTNNB1 and KRAS identified SLC25A22, which encodes mitochondrial glutamate transporter, as a synthetic lethal gene. We investigated the functions of SLC25A22 in CRC cells with mutations in KRAS. We measured levels of SLC25A22 messenger RNA and protein in paired tumor and nontumor colon tissues collected from 130 patients in Hong Kong and 17 patients in China and compared protein levels with patient survival times. Expression of SLC25A22 was knocked down in KRAS mutant CRC cell lines (DLD1, HCT116, LOVO, SW480, SW620, and SW1116) and CRC cell lines without mutations in KRAS (CACO-2, COLO205, HT29, and SW48); cells were analyzed for colony formation, proliferation, glutaminolysis and aspartate synthesis, and apoptosis in Matrigel and polymerase chain reaction array analyses. DLD1 and HCT116 cells with SLC25A22 knockdown were grown as xenograft tumors in nude mice; tumor growth and metastasis were measured. SLC25A22 was expressed ectopically in HCT116 cells, which were analyzed invitro and grown as xenograft tumors in nude mice. Levels of SLC25A22 messenger RNA and protein were increased in colorectal tumor tissues compared with matched nontumor colon tissues; increased protein levels were associated with shorter survival times of patients (P= .01). Knockdown of SLC25A22 in KRAS mutant CRC cells reduced their proliferation, migration, and invasion invitro, and tumor formation and metastasis in mice, compared with cells without SLC25A22 knockdown. Knockdown of SLC25A22 reduced aspartate biosynthesis, leading to apoptosis, decreased cell proliferation in KRAS mutant CRC cells. Incubation of KRAS mutant CRC cells with knockdown of SLC25A22 with aspartate increased proliferation and reduced apoptosis, which required GOT1, indicating that oxaloacetate is required for cell survival. Decreased levels of oxaloacetate in cells with knockdown of SLC25A22 reduced regeneration of oxidized nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. Reduced oxidized nicotinamide adenine dinucleotide inhibited glycolysis and decreased levels of adenosine triphosphate, which inactivated mitogen-activated protein kinase kinase and extracellular signal-regulated kinase signaling via activation of AMP-activated protein kinase. An increased ratio of oxidized nicotinamide adenine dinucleotide phosphate to reduced nicotinamide adenine dinucleotide phosphate induced oxidative stress and glutathione oxidation, which suppressed cell proliferation. Asparagine synthetase mediated synthesis ofasparagine from aspartate to promote cell migration. SLC25A22 promotes proliferation and migration of CRC cells with mutations KRAS, and formation and metastasis of CRC xenograft tumors in mice. Patients with colorectal tumors that express increased levels of SLC25A22 have shorter survival times than patients whose tumors have lower levels. SLC25A22 induces intracellular synthesis of aspartate, activation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase signaling and reduces oxidative stress.

Heterogeneous EGFR gene copy number increase is common in colorectal cancer and defines response to anti-EGFR therapy.

Anti-EGFR therapy is commonly used to treat colorectal cancer (CRC), although only a subset of patients benefit from the treatment. While KRAS mutation predicts non-responsiveness, positive predictive markers are not in clinical practice. We previously showed that immunohistochemistry (IHC)-guided EGFR gene copy number (GCN) analysis may identify CRC patients benefiting from anti-EGFR treatment. Here we tested the predictive value of such analysis in chemorefractory metastatic CRC, elucidated EGFR GCN heterogeneity within the tumors, and evaluated the association between EGFR GCN, KRAS status, and anti-EGFR antibody response in CRC cell lines. The chemorefractory patient cohort consisted of 54 KRAS wild-type (WT) metastatic CRC patients. EGFR GCN status was analyzed by silver in situ hybridization using a cut-off value of 4.0 EGFR gene copies/cell. KRAS-WT and KRAS mutant CRC cell lines with different EGFR GCN were used in in vitro studies. The chemorefractory CRC tumors with EGFR GCN increase (≥4.0) responded better to anti-EGFR therapy than EGFR GCN (<4.0) tumors (clinical benefit, P = 0.0004; PFS, HR = 0.23, 95% CI 0.12–0.46). EGFR GCN counted using EGFR IHC guidance was significantly higher than the value from randomly selected areas verifying intratumoral EGFR GCN heterogeneity. In CRC cell lines, EGFR GCN correlated with EGFR expression. Best anti-EGFR response was seen with KRAS-WT, EGFR GCN = 4 cells and poorest response with KRAS-WT, EGFR GCN = 2 cells. Anti-EGFR response was associated with AKT and ERK1/2 phosphorylation, which was effectively inhibited only in cells with KRAS-WT and increased EGFR GCN. In conclusion, IHC-guided EGFR GCN is a promising predictor of anti-EGFR treatment efficacy in chemorefractory CRC.

Preclinical Activity of the Rational Combination of Selumetinib (AZD6244) in Combination with Vorinostat in KRAS-Mutant Colorectal Cancer Models

Despite the availability of several active combination regimens for advanced colorectal cancer (CRC), the 5-year survival rate remains poor at less than 10%, supporting the development of novel therapeutic approaches. In this study, we focused on the preclinical assessment of a rationally based combination against KRAS-mutated CRC by testing the combination of the MEK inhibitor, selumetinib, and vorinostat, a histone deacetylase (HDAC) inhibitor. Transcriptional profiling and gene set enrichment analysis (baseline and posttreatment) of CRC cell lines provided the rationale for the combination. The activity of selumetinib and vorinostat against the KRAS-mutant SW620 and SW480 CRC cell lines was studied in vitro and in vivo. The effects of this combination on tumor phenotype were assessed using monolayer and 3-dimensional cultures, flow cytometry, apoptosis, and cell migration. In vivo, tumor growth inhibition, (18)F-fluoro-deoxy-glucose positron emission tomography (FDG-PET), and proton nuclear magnetic resonance were carried out to evaluate the growth inhibitory and metabolic responses, respectively, in CRC xenografts. In vitro, treatment with selumetinib and vorinostat resulted in a synergistic inhibition of proliferation and spheroid formation in both CRC cell lines. This inhibition was associated with an increase in apoptosis, cell-cycle arrest in G(1), and reduced cellular migration and VEGF-A secretion. In vivo, the combination resulted in additive tumor growth inhibition. The metabolic response to selumetinib and vorinostat consisted of significant inhibition of membrane phospholipids; no significant changes in glucose uptake or metabolism were observed in any of the treatment groups. These data indicate that the rationally based combination of the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, selumetinib, with the HDAC inhibitor vorinostat results in synergistic antiproliferative activity against KRAS-mutant CRC cell lines in vitro. In vivo, the combination showed additive effects that were associated with metabolic changes in phospholipid turnover, but not on FDG-PET, indicating that the former is a more sensitive endpoint of the combination effects.

Combination of ATP-competitive mammalian target of rapamycin inhibitors with standard chemotherapy for colorectal cancer

ATP-competitive mammalian target of rapamycin (mTOR) inhibitors are in early phase clinical trials. These novel targeted agents, including PP242, are mechanistically distinct from the allosteric, partial mTOR inhibitor, rapamycin. The goal of this study was to evaluate how PP242 best combines with standard chemotherapies for colorectal cancer (CRC), and which subsets of patients are most likely to benefit. The combination index for PP242 plus 5-fluorouracil, oxaliplatin, or irinotecan was determined in CRC cell lines with different mutational backgrounds. In KRAS mutant CRC cell lines, sensitivity to PP242 increases with co-mutation of PIK3CA. Mutation of p53 predicts resistance to chemotherapy, but not PP242. Efficacy of PP242 was comparable to that of standard chemotherapies over the dose range tested. Sensitivity or resistance to PP242 dictates relative synergy or antagonism, respectively, when PP242 is combined with 5-fluorouracil. The same trend exists for PP242 + oxaliplatin, but with a narrower dynamic range. Conversely potency of PP242 and the combination index for PP242 + irinotecan were unrelated, but synergy exists across all dose levels in PP242 and irinotecan sensitive, p53 wild-type cell lines. Overall, our in vitro analysis predicts that mutational status can be used to rank sensitivity to PP242 and standard chemotherapies. Single agent potency can in turn be used to predict the combination index in a drug-specific manner. Our data suggest a clinical trial to determine whether ATP-competitive mTOR inhibitors provide benefit in combination with standard chemotherapies for patients with PIK3CA mutant metastatic CRC, stratified by the presence or absence of KRAS co-mutation.

Abstract C155: Inhibition of TAK1 promotes apoptosis in KRAS and Wnt pathway codependent colon cancers.

Abstract Colorectal cancers frequently harbor KRAS mutations but KRAS-mutant cancers are refractory to conventional and targeted therapeutics. The primary aim of this study was to identify novel therapeutic strategies to target KRAS-mutant colon cancers. Firstly, we found that a subset of KRAS-mutant colorectal cancer cell lines are dependent upon KRAS signaling for survival. Secondly, we found that KRAS-dependent colorectal cancer cells require the TAK1 kinase (MAP3K7) for sustained viability. TAK1 depletion or the small molecule TAK1 kinase inhibitor 5Z-7-oxozeaenol results in profound apoptotic cell death in KRAS-dependent cells. Lastly, we noted that sensitivity to TAK1 disruption is associated with enhanced Wnt signaling resulting from APC mutations combined with KRAS hyperactivation. This effect can be attributed in part to a KRAS-mediated activation of BMP signaling, leading to TAK1 activation. TAK1 then directly regulates Wnt signaling specifically in KRAS-dependent cells. In primary human colorectal cancers, expression of a gene signature derived from TAK1-dependent cell lines is correlated with APC and KRAS mutational status. Thus, our studies reveal TAK1 inhibition as a potential therapeutic strategy for the generally treatment-refractory subset of colorectal cancers that are driven by the KRAS and Wnt pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C155.

Evaluation of antiproliferative and apoptotic effects of the rational combination of the MEK1/2 inhibitor selumetinib (AZD6244) and inhibitors of the hedgehog pathway in colorectal cancer (CRC) cell lines.

422 Background: The MAPK pathway is a crucial regulator of cell proliferation, survival, and resistance to apoptosis. Hyperactivation of this pathway due to mutations in KRAS have been reported in up to 50% of CRC cases. Clinical trials have shown that KRAS patients do not benefit from therapies targeting EGFR, highlighting the need for new therapeutic options. Utilizing differential gene array analyses, we have identified the hedgehog (HH) signaling pathway as a potential mediator of resistance to AZD6244. Based on these results, we tested the rational combination of selumetinib and the HH inhibitor, cyclopamine against human CRC cell lines. Methods: CRC cell lines were exposed to varying concentrations of selumetinib and cyclopamine. For AZD6244, cell lines with IC50≤ 0.1 μM were considered extremely sensitive (ES) and those with IC50≥ 1μM were deemed extremely resistant (ER). Four KRAS mutant cell lines (2ES, 2ER) were selected for combination studies. The antiproliferative effects were assessed using the sulforhodamine B (SRB) cell viability assay, and potential synergy was evaluated using the Chou and Talalay method. Apoptosis was analyzed using bioluminescent caspase 3/7 detection. Results: In all four cell lines tested, synergistic antiproliferative effects of selumetinib and cyclopamine were observed, including resistant lines to selumetinib. We observed significant induction of apoptosis when cell lines were exposed to the combination treatment, independent of their responsiveness to selumetinib in the SRB assay. Conclusions: Treatment of KRAS mutant CRC cell lines with selumetinib and cyclopamine resulted in synergistic inhibition of proliferation, regardless of sensitivity to selumetinib. Interestingly, a significant increase in apoptosis was observed in response to the combination, which may explain the synergy observed by the combination index (CI). In vivo analyses of this combination in cell lines and human CRC explants are ongoing to further validate these results. These preclinical data may suggest a rational combination strategy for patients with KRAS mutant CRC. No significant financial relationships to disclose.