Epigenetics and miRNA as predictive markers and targets for lung cancer chemotherapy

Lung cancer cells show inherent and acquired resistance to chemotherapy. The lack of good predictive markers/novel targets and the incomplete understanding of the mechanisms of resistance limit the success of lung cancer response to chemotherapy. In the present study, we used an isogenic pair of lung adenocarcinoma cell lines; A549 (wild-type) and A549DOX11 (doxorubicin resistant) to study the role of epigenetics and miRNA in resistance/response of non-small cell lung cancer (NSCLC) cells to doxorubicin. Our results demonstrate differential expression of epigenetic markers whereby the level of HDACs 1, 2, 3 and4, DNA methyltransferase, acetylated H2B and acetylated H3 were lower in A549DOX11 compared to A549 cells. Fourteen miRNAs were dys-regulated in A549DOX11 cells compared to A549 cells, of these 14 miRNAs, 4 (has-mir-1973, 494, 4286 and 29b-3p) have shown 2.99 – 4.44 fold increase in their expression. This was associated with reduced apoptosis and higher resistance of A549DOX11cells to doxorubicin and etoposide. Sequential treatment with the epigenetic modifiers trichostatin A or 5-aza-2''-deoxycytidine followed by doxorubicin resulted in: (i) enhanced sensitivity of both cell lines to doxorubicin especially at low concentrations, (ii) enhanced doxorubicin-induced DNA damage in both cell lines, (iii) dysregulation of some miRNAs in A549 cells. In conclusion, A549DOX11 cells resistant to DNA damaging drugs have epigenetic profile and miRNA expression different from the sensitive cells. Moreover, epigenetic modifiers may reverse the resistance of certain NSCLC cells to DNA damaging agents by enhancing induction of DNA damage. This may open the door for using epigenetic profile/miRNA expression of some cancer cells as resistance markers/targets to improve response of resistant cells to doxorubicin and for the use of combination doxorubicin/epigenetic modifiers to reduce doxorubicin toxicity.     

Osteopontin regulates human glioma cell invasiveness and tumor growth in mice

Human malignant glioma cells are characterized by local invasion. In the present study, we investigated the role of osteopontin (OPN) in the invasiveness of human glioma cells isolated from grade IV tumors. We found that the expression levels of OPN in these cell lines paralleled matrix metalloproteinase-2 (MMP-2) expression and cell invasiveness potential. When U87MG glioma cells (with a high-OPN expression level) were stably transformed with specific small hairpin RNA to knock down OPN expression, MMP-2 secretion, cell invasiveness, and tumor growth in implanted brains were dramatically reduced. Conversely, forced expression of OPN in GBM-SKH glioma cells (which expressed OPN at a low level) increased MMP-2 secretion, enhanced cell invasiveness, and increased tumor growth in a rodent xenograft model. Expression of OPN was associated with increased expression of vimentin and decreased expression of glial fibrillary acidic protein. Treatment of glioma cells with 5-aza-2′-deoxycytidine (5-aza-dC) suppressed OPN expression in a concentration-dependent manner. Suppression of OPN expression by 5-aza-dC was associated with reductions in MMP-2 secretion, vimentin expression, cell invasion, intravasation, and tumor growth. These data suggest that OPN may play important roles in regulating cell invasion in glioma cells and that 5-aza-dC may serve as a therapeutic agent for human gliomas.     

A randomised,phase II trial of the DNA-hypomethylating agent 5-aza-2′-deoxycytidine (decitabine) in combination with carboplatin vs carboplatin alone in patients with recurrent,partially platinum-sensitive ovarian cancer

Background:

Our previous laboratory and clinical data suggested that one mechanism underlying the development of platinum resistance in ovarian cancer is the acquisition of DNA methylation. We therefore tested the hypothesis that the DNA hypomethylating agent 5-aza-2′-deoxycytodine (decitabine) can reverse resistance to carboplatin in women with relapsed ovarian cancer.

Methods:

Patients progressing 6–12 months after previous platinum therapy were randomised to decitabine on day 1 and carboplatin (AUC 6) on day 8, every 28 days or carboplatin alone. The primary objective was response rate in patients with methylated hMLH1 tumour DNA in plasma.

Results:

After a pre-defined interim analysis, the study closed due to lack of efficacy and poor treatment deliverability in 15 patients treated with the combination. Responses by GCIG criteria were 9 out of 14 vs 3 out of 15 and by RECIST were 6 out of 13 vs 1 out of 12 for carboplatin and carboplatin/decitabine, respectively. Grade 3/4 neutropenia was more common with the combination (60% vs 15.4%) as was G2/3 carboplatin hypersensitivity (47% vs 21%).

Conclusions:

With this schedule, the addition of decitabine appears to reduce rather than increase the efficacy of carboplatin in partially platinum-sensitive ovarian cancer and is difficult to deliver. Patient-selection strategies, different schedules and other demethylating agents should be considered in future combination studies.     

Anticancer efficacy of cisplatin and trichostatin A or 5-aza-2′-deoxycytidine on ovarian cancer

Background:

To evaluate the anticancer efficacy of the combination of epigenetic modifiers and cisplatin in human ovarian cancer.

Methods:

The effect of trichostatin A (TSA) and 5-aza-2′-deoxycytidine alone or in combination with low-dose cisplatin was evaluated on human ovarian cancer cell lines in vitro. We measured drug interaction by MTS assay, migration by transwell assay, expression of epithelial to mesenchymal transition (EMT) markers (Twist, Snail, Slug, E-cadherin, and N-cadherin), pluripotency markers (Oct4, Sox2, and Nanog), and epigenetic markers (DNMT3A, LSD1 and H3K4me2, H3K4me3, H3K9me2, and H3K9me3) by western blot, and the impact on and characteristics of spheroid growth when exposed to these drugs. Mouse xenografts were used to evaluate the anticancer effect of sequential drug treatment.

Results:

Combination treatment had greater efficacy than single drugs and significantly suppressed cell viability, migration, and spheroid formation and growth. Sequential treatment of cisplatin (1 mg kg⁻¹) followed by TSA (0.3 mg kg⁻¹) significantly suppressed tumorigenicity of HEY xenografts through inhibition of EMT and decreased pluripotency of ovarian cancer cells.

Conclusion:

Epigenetic modifiers potentiate the anticancer efficacy of low-dose cisplatin in ovarian cancer through regulation of EMT and pluripotency, and may provide a promising treatment for ovarian cancer patients.     

Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2′-deoxycytidine in human leukemic KG1 cells

5-azacytidine and 5-aza-2′-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2′-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2′-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2′-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2′-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.     

Cancer testis antigens expression in mesothelioma: role of DNA methylation and bioimmunotherapeutic implications

Recent evidences suggest that malignant mesothelioma may be sensitive to immunotherapy; however, little is known about malignant mesothelioma-associated tumour antigens. Focusing on cancer/testis antigens, the expression of well-characterised immunogenic tumour-associated antigens was investigated in malignant mesothelioma cells. At variance with MAGE-4 and NY-ESO-1, malignant mesothelioma cells frequently expressed MAGE-1, -2 and -3, GAGE 1-2, GAGE 1-6, SSX-2 and SSX 1-5, and distinct malignant mesothelioma cells concomitantly expressed at least four cancer/testis antigens. Additionally, the tumour-associated antigens RAGE-1 was expressed at high levels in both benign and malignant mesothelial cells. Lastly, treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced and up-regulated the expression of the cancer/testis antigen examined in malignant mesothelioma cells. Overall, these findings strongly suggest that cancer/testis antigens-based immunotherapy may represent a suitable therapeutic approach to malignant mesothelioma, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemo-immunotherapeutic strategies in malignant mesothelioma patients.     

MicroRNA-212 functions as an epigenetic-silenced tumor suppressor involving in tumor metastasis and invasion of gastric cancer through down-regulating PXN expression

Altered expression of paxillin (PXN) is closely linked to the pathogenesis progression, metastasis and prognosis of different malignancies including gastric cancer (GC). Epigenetic silencing of tumor-suppressive microRNAs (miRNAs) is a crucial component of the mechanism underlying activation of oncogenes in tumor. To screen for epigenetically silenced miRNAs which target PXN in GC, we performed bioinformatics algorithms and real-time PCR analysis, and identified miR-212 as the optimum candidate gene. A luciferase reporter gene assay validated that miR-212 directly targets the 3’UTR region of PXN. Importantly, miR-212 levels were inversely correlated with PXN expression in GC cell lines and clinical tumor tissues. The use of miR-212 minics decrease PXN mRNA and protein level in GC cell lines. Moreover, low expression of miR-212 and its promoter hypermethylation were causally related and were associated with aggressive tumor phenotype and adverse prognosis in GC. Restoring mir-212 expression by exogenous mirprecursor molecules transfection or reexpression of endogenous miR-212 treated by 5-aza-2’-deoxycytidine (5-aza) can exert similar effect that reduce GC cells invasion and metastasis abilities in vitro by interacting PXN gene. In addition, 5-aza-induced PXN reduction could be partically blocked by miR-212 inhibitor, resulting in a reversal of weankening cell migration and invasion ability of 5-aza. A rescue experiment and a loss-of-function experiment in vitro and vivo showed that PXN restoration rescues migration and invasion phenotype in miR-212 overexpressed GC cell lines and PXN knockdown blocks GC cells migration and invasion in the presence miR-212 inhibitors. Taken together, our results clearly show that overexpression of PXN induced by methylationsuppressed miR-212 promotes tumor metastasis and invasion, and regulation of miR-212 expression may be a novel therapeutic strategy for gastric cancer.     

Molecular markers in colorectal cancer: genetic bases for a customised treatment

Colorectal cancer (CRC) is the second leading cause of cancer death in Western countries. CRC treatment is based on the employment of three chemotherapeutic drugs, including 5-fluorouracil, oxaliplatin and irinotecan, and the use of recently incorporated targeted agents directed to vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). The approval of these biologicals and of others to come holds great promise for the improvement of patient outcome. The molecular bases for this lethal disease have been extensively investigated, laying the foundations for a rational and customised treatment approach, expanding the therapeutic index of current drugs and easing the incorporation of new molecules. Individual markers have been mainly investigated based on drug targets and metabolism. Also, the increasing availability of highthroughput technologies has prompted the opportunity for blind studies capable of screening new markers and of identifying the specific oncogenic pathways responsible for drug resistance in a given patient. An updated review of the field is presented in this article.     

Reduced expression of intercellular adhesion molecule-1 in ovarian adenocarcinomas.

Ovarian adenocarcinomas develop as the result of multiple genetic and epigenetic changes in the precursor ovarian surface epithelial (OSE) cells which result in a malignant phenotype. We investigated changes in gene expression in ovarian adenocarcinoma using a cDNA array containing 588 known human genes. We found that intercellular adhesion molecule-1 (ICAM-1) was expressed at lower levels in the ovarian tumour cell lines OAW42, PEO1 and JAM than in the immortalised human ovarian surface epithelial cell line HOSE 17.1. Further investigation revealed ICAM-1 was expressed in the surface epithelium of normal ovaries and both mRNA and protein expression levels were reduced in the majority of ovarian adenocarcinoma cell lines and primary tumours. ICAM-1 expression was increased in 8/8 cell lines treated with the de novo methyltransferase inhibitor 5-aza-2'-deoxycytidine, indicating that methylation of CpG islands may play a role in the down-regulation of its expression in primary tumours. There was a significant association between patients whose tumours expressed ICAM-1 and survival (P = 0.03), suggesting that expression levels of ICAM-1 may have clinical relevance.     

Synergy of 5-aza-2′-deoxycytidine (DAC) and paclitaxel in both androgen-dependent and -independent prostate cancer cell lines

To determine the synergy of 5-aza-2′-deoxycytidine (DAC) and paclitaxel (PTX) against prostate carcinoma (PC) cells by isobolographic analysis. We demonstrated that DAC could significantly increase the susceptibility of PC cells to PTX, and confirmed the synergy of DAC and PTX. DAC enhanced the PTX induced up-regulation of caspase activity and antiproliferative effect, resulting in an increase of cells in subG1 and G2/M phases. In addition, the synergy was observed in both androgen-dependent and -independent PC cell lines. It suggested that combination chemotherapy with DAC and PTX might be a new strategy to improve the clinical response rate of PC.     

RARRES3 suppressed metastasis through suppression of MTDH to regulate epithelial-mesenchymal transition in colorectal cancer

It has been reported that Retinoic acid receptor responder 3 (RARRES3) could suppress the metastasis of colorectal cancer (CRC). However, the underlying mechanism by which RARRES3 suppresses metastasis remains unknown. To investigate the functional involvement of RARRES3 in CRC, we first analyzed the expression of this protein between human CRC clinical samples and their corresponding normal controls and tested its correlation with clinicopathology as well as prognosis of CRC. We also examined the endogenous expression of RARRES3 by western-blot in a panel of CRC cell lines with different metastatic capacity. Cell proliferation, migration and invation of the CRC cell lines with either knockdown or reexpression of RARRES3 were examined by MTT, transwell and wound healing assays, respectively. The intrecellular signaling pathways affected by manipulations of RARRES3 in CRC cells were determined by western blot. Immunoprecipitation (IP) was employed to assess the interactionbetween proteins. To investigate the metastatic ability in vivo, CRC cell lines with manipulations of RARRES3 expression were inoculated in nude mice through tail vein injection. We confirmed that RARRES3 was significantly down-regulated in CRC tissues compared with normal controls. RARRES3 expression was not correlated with prognosis but significantly associated with CRC differentiation and lymphnodes metastases. We also found that RARRES3 was able to significantly suppress the metastasis of CRC cells both in vitro and in vivo through the regulation of epithelial-mesenchymal transition (EMT) process during which RARRES3 interactedwith MTDH in an opposite way. Taken together, we for the first time found that RARRES3 was able to suppress the metastasis of CRC both in vitro and in vivo via suppression of MTDH so as to regulate EMT.     

Caveolin-1 is down-regulated in alveolar rhabdomyosarcomas and negatively regulates tumor growth

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with histological variant of rhabdomyosarcoma known as alveolar rhabdomyosarcoma (ARMS) have a 5-year survival of less than 30%. Caveolin-1 (CAV1), encoding the structural component of cellular caveolae, is a suggested tumor suppressor gene involved in cell signaling. In the present study we report that compared to other forms of rhabdomyosarcoma (RMS) CAV1 expression is either undetectable or very low in ARMS cell lines and tumor samples. DNA methylation analysis of the promoter region and azacytidine-induced re-expression suggest the involvement of epigenetic mechanisms in the silencing of CAV1. Reintroduction of CAV1 in three of these cell lines impairs their clonogenic capacity and promotes features of muscular differentiation. In vitro, CAV1-expressing cells show high expression of Caveolin-3 (CAV3), a muscular differentiation marker. Blockade of MAPK signaling is also observed. In vivo, CAV1-expressing xenografts show growth delay, features of muscular differentiation and increased cell death. In summary, our results suggest that CAV1 could function as a potent tumor suppressor in ARMS tumors. Inhibition of CAV1 function therefore, could contribute to aberrant cell proliferation, leading to ARMS development.     

5-氮杂-2′-脱氧胞苷对非小细胞肺癌体外生长及侵袭能力和TFPI-2基因mRNA表达的影响

目的 探讨5-氮杂-2′-脱氧胞苷(5-aza-2-deoxycytidine,5-Aza-CdR)干预对非小细胞肺癌(nonsmallcell non cancer, NSCLC)细胞株A549生长增殖及其组织因子途径抑制物2(tissue factor pathwayinhibitor-2,TFPI-2)基因表达的影响,同时探讨5-Aza-CdR能否通过恢复TFPI-2基因表达抑制非小细胞肺癌A549细胞侵袭能力。方法 用不同浓度的5-Aza-CdR处理A549细胞株,MTT法检测药物处理24、48、72 h后的细胞增殖活性,流式细胞仪(fl ow cytometry, FCM)法检测药物处理72 h后细胞周期分布,Real-time PCR技术检测药物处理72h后A549细胞TFPI-2基因mRNA的表达,Transwell小室法测定药物处理24h后A549细胞体外侵袭能力的变化。 结果 MTT检测显示不同浓度5-Aza-CdR处理A549细胞24、48、72h后,细胞的生长受到抑制,且抑制作用呈明显的剂量和时间依赖性。FCM检测分析显示0、1、5、10 μM 5-Aza-CdR处理A549细胞72 h 后,细胞增殖指数逐渐降低,分别为（30.43±0.99)%、（23.89±0.83)%、（16.19±0.34)%、（6.49±0.55%（P＜0.05）。Real-timePCR检测显示0、1、5、10 μM的5-Aza-CdR处理A549细胞72 h后,相对mRNA表达水平分别为（1±0)、（1.49±0.14)、（1.86±0.09)、（5.80±0.15)（P<0.05）,TFPI-2 基因mRNA表达呈明显的上升趋势,且随着药物浓度增加而增加。Transwell小室法检测显示每一高倍镜下平均穿膜细胞数分别为（316.15±18.7)、（84.15±12.14)、（28.85±7.13)、（14.35±3.33),均明显低于对照细胞（P<0.05）。结论 5-Aza-CdR能通过降低TFPI-2基因的甲基化而恢复其在非小细胞肺癌细胞株A549细胞中的表达,并抑制A549细胞的增殖和侵袭。     

Increased cytotoxicity and bystander effect of 5-fluorouracil and 5-deoxy-5-fluorouridine in human colorectal cancer cells transfected with thymidine phosphorylase.

5-Fluorouracil (5-FU) and 5'-deoxy-5-fluorouridine (5'-DFUR), a prodrug of 5-FU, are anticancer agents activated by thymidine phosphorylase (TP). Transfecting the human TP cDNA into cancer cells in order to sensitize them to these pyrimidine antimetabolites may be an important approach in human cancer gene therapy research. In this study, an expression vector containing the human TP cDNA (pcTP5) was transfected into LS174T human colon carcinoma cells. Eight stable transfectants were randomly selected and analysed. The cytotoxic effects of 5-FU and 5'-DFUR were higher in TP-transfected cells as compared to wild-type cells. The maximal decreases in the IC50 were 80-fold for 5-FU and 40-fold for 5'-DFUR. The increase in sensitivity to these pyrimidines of TP-transfected cells significantly correlated with the increase in both TP activity and TP expression. Transfected clone LS174T-c2 but not wild-type cells exhibited formation of [3H]FdUMP from [3H]5-FU. In addition the LS174T-c2 clone enhanced the cytotoxic effect of 5'-DFUR, but also that of 5-FU, towards co-cultured parental cells. For both anti-cancer agents, this bystander effect did not require cell-cell contact. These results show that both 5-FU or 5'-DFUR could be used together with a TP-suicide vector in cancer gene therapy.     

5-氮-2′-脱氧胞苷对人卵巢癌细胞凋亡及DNA错配修复基因表达的影响

目的:观察5-氮-2′-脱氧胞苷(5-aza-2′-deoxycytidine,又称5-aza-CdR)对卵巢癌细胞SKOV3和3AO增殖凋亡及DNA错配基因hMLH1和hMLH2表达的影响。方法:以特异性甲基转移酶抑制剂5-aza-CdR0.5、5、50μmol/L处理人卵巢癌细胞SKOV3和3AO3d,继续常规培养7d后,采用MTT比色法观察细胞经药物处理前后的增殖活性,用流式细胞术分析5-aza-CdR对细胞凋亡影响,以半定量逆转录-聚合酶链反应(RT-PCR)检测细胞经5-aza-CdR处理前后DNA错配修复基因hMLH1和hMSH2mRNA表达水平的改变。结果:人卵巢癌细胞SKOV3和3AO经5-aza-CdR处理后,与对照组比较,0.5、5、50μmol/L均能明显抑制肿瘤细胞生长,随着5-aza-CdR浓度增加,细胞增殖速度下降。SKOV3经5-aza-CdR0.5、5、50μmol/L处理后细胞的凋亡率分别为(10.59±1.57)%、(17.52±1.72)%、(34.10±1.45)%,3A0经0.5、5、50μmol/L5-aza-CdR处理后细胞的凋亡率分别为(11.11±2.21)%、(17.24±1.11)%、(26.53±2.00)%,与对照组相比均有统计学意义(P<0.01);且凋亡率与剂量成正相关(FSKOV3=227.6,PSKOV3<0.01;F3AO=108.4,P3AO<0.01)。经5-aza-CdR处理后的两株卵巢癌细胞中hMLH1和hMLH2的mRNA表达量有不同程度的增加(P<0.01),且与药物存在剂量依赖性。结论:在人卵巢癌细胞株SKOV3和3AO中,5-aza-CdR可部分逆转hMLH1和hMLH2的失活,恢复其生长调控功能,抑制肿瘤细胞生长,并诱导细胞凋亡。     

Lactate dehydrogenase A negatively regulated by miRNAs promotes aerobic glycolysis and is increased in colorectal cancer

Reprogramming metabolism of tumor cells is a hallmark of cancer. Lactate dehydrogenase A (LDHA) is frequently overexpressed in tumor cells. Previous studies has shown higher levels of LDHA is related with colorectal cancer (CRC), but its role in tumor maintenance and underlying molecular mechanisms has not been established. Here, we investigated miRNAs-induced changes in LDHA expression. We reported that colorectal cancer express higher levels of LDHA compared with adjacent normal tissue. Knockdown of LDHA resulted in decreased lactate and ATP production, and glucose uptake. Colorectal cancer cells with knockdown of LDHA had much slower growth rate than control cells. Furthermore, we found that miR-34a, miR-34c, miR-369-3p, miR-374a, and miR-4524a/b target LDHA and regulate glycolysis in cancer cells. There is a negative correlation between these miRNAs and LDHA expression in colorectal cancer tissues. More importantly, we identified a genetic loci newly associated with increased colorectal cancer progression, rs18407893 at 11p15.4 (in 3′-UTR of LDHA), which maps to the seed sequence recognized by miR-374a. Cancer cells overexpressed miR-374a has decreased levels of LDHA compared with miR-374a-MUT (rs18407893 at 11p15.4). Taken together, these novel findings provide more therapeutic approaches to the Warburg effect and therapeutic targets of cancer energy metabolism.     

Identification of BIK as an unfavorable prognostic marker and novel therapeutic target in microsatellite stable colorectal cancer harboring KRAS mutations

KRAS mutations lead to persistent activation of multiple downstream effectors that drive the cancer phenotype. Approximately 30%-50% of colorectal cancer (CRC) patients harbor KRAS mutations, which confer more aggressive tumor biology and shorter overall survival (OS), especially in microsatellite stable (MSS) metastatic CRC. Given that KRAS mutant protein has been proven difficult to target directly, identifying genes that function closely with KRAS and targeting these genes seems to be a promising therapeutic strategy for KRAS-mutated MSS CRC. Here, KRAS function-sensitive genes were identified by assessing the correlation between gene dependency scores from CRISPR knockout screens and KRAS mRNA expression in KRAS-mutated MSS CRC cell lines in the Cancer Cell Line Encyclopedia (CCLE) database. If the correlation coefficient was ≥ 0.6, the gene was considered a KRAS function-sensitive gene. Then KRAS function-sensitive genes related to prognosis were screened out in The Cancer Genome Atlas (TCGA) cohort, and the prognostic value was validated in the Gene Expression Omnibus (GEO) cohort. Single-sample gene set enrichment analysis (ssGSEA) was performed to investigate the potential mechanisms. PockDrug-Server was used to predict the druggability of candidate genes. The results showed that in 20 KRAS-mutated MSS CRC cell lines, 13 genes were identified as KRAS function-sensitive genes. Of these 13 genes, only BIK expression was significantly associated with progression-free survival (PFS) and OS, and the BIK-high patients had significantly poorer PFS (HR=3.18, P=0.020) and OS (HR=4.74, P=0.030) than the BIK-low patients. Multivariate Cox regression analysis revealed high BIK expression as an independent predictor for poorer prognosis in KRAS-mutated MSS CRC. The prognostic value of BIK was also successfully validated in a GEO cohort. The results of ssGSEA showed that the BIK-high group was more prone to strong metastasis activity than the BIK-low group. Pocket druggability prediction analysis presented that BIK had three druggable pockets, and their druggability scores were above 0.8. These findings suggested that BIK is a promising prognostic marker and therapeutic target in KRAS-mutated MSS CRC.