KRAS G12C mutation‐induced TOPK overexpression contributes to tumour progression in non‐small cell lung cancer

Abstract KRAS mutation is the most frequent type of genetic mutation in non‐small cell lung cancer (NSCLC), especially in lung adenocarcinoma. However, KRAS mutation can affect many biological processes and the mechanisms underlying KRAS mutation‐mediate carcinogenesis in NSCLC have not been fully understood. In this research, we found that KRAS G12C mutation was associated with the upregulation of T‐LAK cell‐originated protein kinase (TOPK), which is a well‐known serine/threonine MAPK‐like protein kinase implicated in tumorigenesis. The overexpression of TOPK significantly promoted the malignant phenotype of A549 cells, and TOPK silencing impaired the malignant phenotype with KRAS G12C mutation. Moreover, we demonstrated that TOPK level was regulated by MAPK/ERK signalling and the transcription factor Elk1. TOPK was also found to promote the activation of NF‐κB signalling in A549 cells with KRAS G12C mutation via facilitating the phosphorylation of TAK1. In the in vivo tumorigenesis model, the administration of TOPK inhibitor OTS514 enhanced the anticancer effect of 5‐FU, and the combinatory use of OTS514 and KRAS G12C inhibitor AMG510 showed synergistic anti‐tumour effect. These results suggest that KRAS‐TOPK axis contributes to the progression of NSCLC and targeting this axis could synergize with anticancer effect of the existing chemotherapeutics.


| INTRODUC TI ON
As one of the most frequent type of malignant tumours, lung cancer poses a serious health threat to public health worldwide. 1 According to histopathological classification, lung cancers are classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), and NSCLC accounts for about 85% of the total diagnosed cases of lung cancer. 2 For patients in early stage, surgical resection is the optimal approach for the treatment, and chemotherapy remains as the mainstay treatment for advanced NSCLC. 3 However, the 5-year survival rate of NSCLC is only 17.5%. 4 Therefore, exploring the pathogenesis and molecular mechanisms underlying the progression of NSCLC is crucial to develop targeted therapies for more effective treatment of NSCLC.
The dysregulated cell proliferation in cancer cells is mainly caused by the accumulation of genetic mutations. Kirsten rat sarcoma viral oncogene homologue (KRAS) mutation is one of the most prevalent genetic mutations in NSCLC, and its activation has been described in approximately 35% cases of NSCLC. 5 Wild type KRas protein cycles between two different states: GDP-bound inactive state (KRas-GDP) and GTP-bound active state (KRas-GTP). The prevalent mutations including G12, G13, and Q61 lock the mutated KRas protein in a constitutively active GTP-bound state, leading to a persistent oncogenic signalling. 6 Glycine-to-cysteine (G12C) mutation is a dominant KRAS mutation accounting for nearly 44% of KRAS mutations in NSCLC patients, which has been found to be extremely common in NSCLC patients with smoking history. 7 Recently, AMG510 has been developed as the first KRAS G12C inhibitor, and it shows strong anti-cancer efficiency and suppresses the progression of NSCLC. 8 These findings suggest that researches that targeting KRAS G12C mutation can serve as a promising strategy in the treatment of NSCLC.
Recently, T-LAK cell-originated protein kinase (TOPK), also known as PDZ-binding-kinase (PBK), has been reported as a critical oncogenic protein in multiple cancers. 9-12 TOPK has been reported to be upregulated in a variety of cancers, such as oral cancer, breast cancer, leukaemia and prostate cancer, [9][10][11][12] whereas its expression is relatively low in normal tissues. TOPK is implicated in various physiological and biological processes, such as autophagy, apoptosis, and cell proliferation. [13][14][15] In lung cancer, the upregulation of TOPK was reported to promote chemical-resistance towards EGFR tyrosine kinase inhibitors, 16 and facilitate hypoxia-induced epithelialmesenchymal transition and the invasion of NSCLC cells. 17 However, the molecular mechanism underlying TOPK upregulation in NSCLC remained to be elucidated.
In the current study, we explored the expression of TOPK in lung tissues of NSCLC patients with KRAS G12C mutation. We found that TOPK expression was significantly higher in tissues with KRAS G12C mutation compared with those with wild type KRAS gene. We further showed that TOPK was induced by the excessive activation of transcription factor Elk1 through MAPK/ERK signalling.
Furthermore, TOPK facilitated the activation of NF-κB signalling via promoting the phosphorylation of TAK1 in NSCLC cells. The administration of TOPK inhibitor OTS514 enhanced the anticancer effect of 5-FU in the in vivo tumorigenesis model, and the combinatory use of OTS514 and KRAS G12C inhibitor AMG510 showed synergistic anti-tumour effect. In summary, these results suggest that the constitutive activation of KRAS-TOPK axis contributes to the malignant progression of NSCLC, and targeting this axis could serve as a targeted therapy for NSCLC treatment. genome version (with the parameters "-no-novel-junctions", "nocoverage-search option", "library-type = fr-firststrand" and "-G" when specifying the genome file). The overall alignment rates were ~99%. Cufflinks v2.2.1 (with the -G parameter and the same GTF from tophat analysis) was used to derive expression (FPKM) values from the alignments using the same genome/annotation. Cuffmerge package (with the -g and -s parameters, the same GTF and the reference FASTA) was then used to merge the resulting transcripts before performing differential gene expression by Cuffdiff package (using the -b parameter with reference FASTA, and with the --max-bundle-frags 5,000,000 and -u parameters). FDR-adjusted p value after Benjamini-Hochberg correction for multiple-testing were used as the statistics to define the differential expression.
Genes with FDR-adjusted p < 0.05 are considered to be differentially expressed.

| Cellcultureandstablecelllinegeneration bylentivirus
Human NSCLC cell line A549 was purchased from Type Culture Collection of Chinese Academy of Sciences. Cells were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS; Gibco), 100 U/ml of penicillin and 100 μg/ml of streptomycin in a humidified incubator containing 5% CO 2 at 37°C. The A549 cell lines with stable expression of KRAS WT or KRAS G12C were generated as previous described. 18 The silencing and overexpression lentiviruses of TOPK were synthesized by Genscript Biotech company. The sequences used for TOPK silencing are as follow: (Mut-KD1: ATT AGT GCA TAC AGA GAA GAG TT),   Mut-KD2: GTC TGT GTC TTG CTA TGG AAT. To generate stable cells   with TOPK silencing or overexpression, cells were infected with the corresponding recombinant lentivirus at a MOI (multiplicity of infection) = 5 in the presence of 10 μg/ml polybrene (Sigma, tr-1003-g).
Infected cells were selected with 1.0 μg/ml puromycin for 2 weeks to eliminate the uninfected cells before further experiment.

| Quantitativerealtime-PCR(qRT-PCR)and luciferasereporterassay
Quantitative real time-PCR was performed as previously reported. 19 TRIzol reagent (Invitrogen) was used for total RNA extraction.

| Dualluciferasereporterassay
The promoter activities of TOPK and NF-κB were examined by dual luciferase reporter assay. The luciferase reporters containing cor-

| Colonyformationassay
Colony formation assay was performed as described. 21

| Apoptosisanalysis
For apoptosis analysis, Annexin V-fluorescein isothiocyanate Apoptosis Detection Kit (BD Biosciences) was used according to the manufacturer's instruction, followed by the analysis by FACSan flow cytometry (BD Biosciences). For TUNEL staining, TUNEL Assay Kit (HRP-DAB, Elasbscince) was used to stain the apoptotic cells, and the images were captured under Leica AM6000 microscope.

| Transwellandmigrationinvasionassay
Cells with different treatments were trypsinized and re-suspended in serum-free medium. The transwell upper chamber coated with Matrigel (BD Biosciences) was used for invasion assay. The transwell chamber without Matrigel coating was used for migration assay. 2.5 × 105 cells were inoculated into the upper chamber in serum-free medium and 500 μl of 10% serum-containing medium was added to the lower chamber. After 24 h, culture medium was discarded and the cells were fixed with 4% paraformaldehyde at room temperature for 10 min and stained with 0.5% crystal violet (Beyotime) for 20 min. Cells were photographed under Leica AM6000 microscope.

| Westernblottinganalysisand immunoprecipitation(IP)
RIPA buffer (Sigma) was used to extract the total protein.
Concentration of protein was measured by BCA protein quantification kit (Thermo Scientific). Western blot and IP were performed as described. 21 The antibodies used in the research were as fol-

| Statisticalanalyses
The data in the study were expressed as means ± SD from at least three independent experiments. Survival of NSCLC patients was described by Kaplan-Meier and analysed by log-rank test. The statistical difference between two groups was compared using unpaired student's t tests. Comparisons among multiple groups were analysed using one-way analysis of variance (ANOVA) with Tukey's post hoc test for pairwise comparison. Comparisons of data at multiple time points were examined using two-way ANOVA. p < 0.05 was considered to be statistically significant.

| TOPKisupregulatedinNSCLCtissuesand cellswithKRAS G12C mutation
To explore the differentially expressed genes between A549 cells with KRAS G12C mutation and KRAS WT , we established A549 cells stably expressing KRAS WT or KRAS G12C . The stable expression of KRAS WT or KRAS G12C was examined by Western blot ( Figure S1A).
Cells were subjected to RNA-seq analysis, and differentially gene expression analysis (fold change of at least 2.0 and adjusted p value of <0.05) showed that TOPK was among the genes upregulated in KRAS G12C A549 cells ( Figure 1A,B, Table S1). To analyse the association of TOPK with the survival of NSCLC patients, we extracted the survival data of a cohort of NSCLC patients from The Cancer Genome Atlas website (TCGA) database. As shown in Figure 1C, Kaplan-Meier survival analysis suggested that a high level of TOPK was associated with a poorer 5-year overall survival (OS) in NSCLC patients, which was consistent with the previous reports about the impaired survival in NSCLC patients with KRAS G12C mutation. 22,23 To confirm the finding, we collected 10 KRAS WT NSCLC and 10 KRAS G12C NSCLC tissues and demonstrated the overexpression of TOPK in KRAS G12C tissues compared with KRAS WT tissues by RT-qPCR ( Figure 1D). We also compared the protein levels of TOPK between normal lung tissues and the NSCLC tissues with KRAS WT or KRAS G12C . Western blot and IHC staining results showed that TOPK was upregulated in all NSCLC tissues when compared to the normal lung tissues, and there was a relatively higher expression in NSCLC tissues KRAS G12C mutation when compared to the NSCLC tissues with KRAS WT tissues ( Figure 1E,F). Consistently, in the A549 cells with KRAS G12C mutation, the TOPK protein level was significantly higher when compared to that of wild type cells ( Figure S1B). Taken together, these findings suggest that the upregulation of TOPK expression is positively correlated with KRAS G12C mutation in NSCLC.

| TOPKisindispensableforthemalignant phenotypeofNSCLCcellswithKRAS G12C mutation
To investigate the functional role of TOPK in NSCLC cells, we ap-

| TOPKexpressionisdependentonthe activityofMAPK/ERKsignallinginNSCLCcells
As KRAS is an upstream regulator of MAPK/ERK pathway, we hypothesized that TOPK expression is under the regulation of MAPK/ ERK signalling pathway. We therefore treated KRAS G12C A549 cells with different inhibitors to determine which pathway is the major contributor to the TOK upregulation. As shown in Figure 3A, VX-702 (p38 inhibitor) suppressed the phosphorylation of p38, but did not affect the phosphorylation level of MEKK1, MKK3 and had no effect on TOPK expression. LY294002, an inhibitor of PI3K, significantly decreased the phosphorylation level of PI3K and AKT, but did not reduce TOPK expression ( Figure 3B). In addition, we detected the relationship between TOPK and MAPK/ERK signalling by using AZ628, which is widely used as a Raf inhibitor. The phosphorylation levels of Raf, MEK1, ERK and Elk1 were decreased upon AZ628 treatment, and TOPK expression level was also sigfnciantly decreased in AZ628 treated A549 cells ( Figure 3C). Taken together, these findings suggest that the expression of TOPK is upregulated is dependent on the activity of MAPK/ERK signalling pathway in NSCLC cells.

| TOPKistranscriptionallyregulatedbyElk1
In Figure 3C, we observed that the phosphorylation level of Elk1 was suppressed and TOPK was downregulated by AZ628 treatment, and Elk1 was well-known as a critical transcription factor downstream of MAPK/ERK signalling pathway. 24 We assumed Results are presented as means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 that TOPK could be transcriptionally regulated by Elk1. We therefore silenced Elk1 by lentivirus, and the knockdown efficiency was shown in Figure 4A. We found that the silencing of Elk1 decreased the expression of TOPK at both protein and mRNA levels ( Figure 4B,C). In A549 cells with KRAS G12C mutation, the phosphorylation levels of Elk1 and ERK and the expression of TOPK were remarkably increased ( Figure 4D,E). Furthermore, we also observed that the expression level of total Elk1 was enhanced by KRAS G12C mutation ( Figure 4D,F), which was consistent with a previous report in colon cancer. 25 In addition, ten putative Elk1binding sites were identified in the 2-kb promoter of TOPK by an online tool JASPAR (http://jaspar.gener eg.net) ( Figure 4G). We therefore cloned the promoter region of TOPK gene locus into a luciferase reporter and performed the dual luciferase activity assay to examine the transcription activity of TOPK promoter.

F I G U R E 3 TOPK expression is regulated by MAPK/ERK signalling in NSCLC cells. (A). KRAS
TOPK promoter activity in KRAS G12C was significantly higher in A549 cells with KRAS G12C mutation when compared to KRAS WT A549 cells ( Figure 4H). Meanwhile, in KRAS WT A549 cells, TOPK promoter activity was increased by Elk1 in a dose-dependent manner ( Figure 4I), and the silencing of Elk1 dramatically suppressed the activity of TOPK promoter in KRAS G12C A549 cells ( Figure 4J).
Taken together, these findings suggest that TOPK was transcriptionally regulated by transcription factor Elk1.

| TOPKpromotesNFκ B signalling activation viaregulatingTAK1phosphorylation
NF-κB signalling pathway is one of the most crucial pathways implicated in the regulation of cell proliferation and tumorigenesis. 26 Therefore we evaluated the activation status of NF-κB signalling in KRAS WT and KRAS G12C A549 cells. As shown in Figure 5A, the phosphorylation levels of p65, IKKβ and IκBα were all increased in KRAS G12C cells. With the administration of OTS514 (a specific inhibitor for TOPK), the phosphorylation levels of p65, IKKβ and IκBα were significantly suppressed ( Figure 5B). In addition, we examined the effect of TOPK on adaptor proteins (such as TRAF2, TRAF6, TAK1 and IKKβ)-induced activation of NF-κB transcription activity using a NF-κB luciferase reporter. NF-κB transcriptional activity was enhanced by TOPK overexpression in KRAS WT A549 cells transfected with TRAF2, TRAF6 and TAK1 expression vector, but TOPK overexpresison did not have effect in IKKβ expression cells, which indicated that TAK1 may be a potential target of TOPK ( Figure 5C). Using the IP assay, we found that TOPK could physically interact with TAK1 in KRAS G12C cells ( Figure 5D). Moreover, the interaction was confirmed by the overexpression of Myc-TOPK and Flag-TAK1 in KRAS WT cells ( Figure 5E). Moreover, we found that that TOPK significantly enhanced the phosphorylation of TAK1 at S412 in a dose-dependent manner ( Figure 5F), and the silencing of TOPK decreased the level of phos-TAK1 ( Figure 5G).
Taken together, these findings suggest that TOPK interacts with TAK1 and promotes TAK1 phosphorylation to activate NF-κB signalling.

5-FU is a widely used chemotherapy agent in NSCLC treatment;
however its application is constrained by the development of drug resistance. 27,28 To test whether TOPK regulates the sensitivity to 5-FU induced anticancer effect, we treated KRAS G12C A549 cells with TOPK inhibitor OTS514, 5-FU or in combination. As shown in Figure 6A, we found that OTS514 and 5-FU could induce apoptosis in KRAS G12C cells, and the combination of OTS514 and 5-FU showed strong synergistic effect in apoptosis induction. A similar result was observed in colony formation assay and CCK-8 proliferation assay ( Figure 6B,C). In addition, the phosphorylation levels of ERK and IκBα were strongly decreased in KRAS G12C cells treated with OTS514 + 5-FU combination ( Figure 6D). Furthermore, we found that the combination of OTS514 + 5-FU induced more apoptotic cells and showed a more profound inhibition on cell proliferation in KRAS G12C cells when compared with KRAS WT cells ( Figure 6E,F). The synergistic effect of OTS514 and 5-FU was also validated in xenograft tumour model ( Figure 6G), and the TUNEL staining in xenograft tumour section showed that the combination of OTS514 and 5-FU administration showed strong synergistic effect in apoptosis induction ( Figure 6H). Taken together, these findings suggest that TOPK inhibitor OTS514 could enhanced the effect of 5-FU against NSCLC with KRAS G12C mutation.

| CombinationofOTS514andAMG510shows anti-tumour effect in KRAS G12C A549cells
AMG510 is the first inhibitory agent that selectively targets KRAS G12C mutation. 8 We further examined the anticancer effect of the combination of OTS514 and AMG510 on NSCLC. As shown in Figure 7A, the highest percentage of apoptosis was found in KRAS G12C cells treated with OTS514 + AMG510 combination. Cell proliferation was also significantly suppressed by the combination of OTS514 and AMG510 ( Figure 7B,C). Consistently, the phosphorylation levels of ERK and IκBα were greatly decreased by the simultaneous treatment of OTS514 and AMG510 ( Figure 7D). In the xenograft tumour model, OTS514 and AMG510 combination also showed strong synergistic effect against tumorigenesis, which was also evidenced by the increased apoptotic events in the tumour section ( Figure 7E,F).
Taken together, these findings suggest that the combinatory use of OTS514 and AMG510 could serve as an anticancer strategy against NSCLC cells with KRAS G12C mutation.

| Evaluationoftheanticancereffectof OTS514,AMG510and5-FUcombinationinKRAS G12C A549cells
To further evaluate the anticancer potential of the combination of OTS514, AMG510 and 5-FU, we treated KRAS G12C A549 cells with different combination of drugs and examined the cell death and proliferation. As expected, we observed that the combinations of 5-FU + OTS514 and 5-FU + AMG510 both showed greater anticancer effect in KRAS G12C A549 cells in comparison to the 5-FU treatment alone (Figure 8A-C). Moreover, when the three agents were applied together (5-FU + OTS514 + AMG510), an even stronger apoptosis induction and anti-proliferation effects were observed ( Figure 8A-C). Therefore, the combinatory usage of 5-FU, OTS514 and AMG510 could potentially produce the optimal anticancer effect against NSCLC with KRAS G12C mutation. . CCK8 proliferation assay in KRAS G12C cells with the above treatments. (C). Colony formation assay in KRAS G12C cells in KRAS G12C cells with the above treatments. Results are presented as means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 axis, 17 and promote the EMT and invasion of breast cancer cells by upregulating TBX3 in TGF-β1/Smad signalling. 10 In addition, TOPK suppresses p53-mediated transcription of pro-apoptotic proteins to inhibit cell death in tumour cells. 34 TOPK can also promote breast cancer cell proliferation by targeting geranylgeranylation signalling. 35 However, the role of TOPK in NSCLC and the relationship between TOPK and KRAS G12C mutation remain largely unknown. In the current study, we reported that TOPK is significantly upregulated in NSCLC tissues and A549 cells with KRAS G12C mutation. Its overexpression is induced mainly by MAPK/ERK signalling and regulated by transcription factor Elk1. Importantly, TOPK overexpression not only supports the malignant phenotype of NSCLC cells in vitro, but also promotes the tumorigenesis of NSCLC cells in mouse model. Furthermore, we also observed that TOPK could activate NF-κB signalling by interacting with TAK1 and phosphorylating TAK1. The enhanced NF-κB signalling could also confer survival advantages in NSCLC cells.
Combination of different chemotherapeutics has been suggested to be effective approaches to enhance anticancer effect, and the proportion of combined drug usage in cancer treatment has increased in recent years. 36,37 In the current research, we found that the combination of OTS514 (TOPK inhibitor) and 5-FU synergistically induces cell apoptosis and strongly suppresses the proliferation of KRAS G12C A549 cells. Moreover, compared to KRAS WT cells, KRAS G12C cells seem to be more sensitive for the combined treatment of OTS514 and 5-FU.
Furthermore, the combination of OTS514 and AMG510 (KRAS G12C mutant inhibitor) also shows strong synergistic effect against NSCLC cells with KRAS G12C mutation. When the three agents were applied together, the effect was further boosted with a profound effect in apoptosis induction and cell growth inhibition. These results together indicate that as a downstream of KRAS, TOPK is highly expressed in NSCLC cells with KRAS G12C mutation. The combinatory targeting of KRAS mutation and TOPK in NSCLC cells could be of great significance for overcoming the drug resistance of 5-FU, which needs to be further evaluated in clinical practice.
In conclusion, we reported the regulation of TOPK in NSCLC

FU N D I N GI N FO R M ATI O N
None.

CO N FLI C TO FI NTE R E S TS TATE M E NT
The authors declare no competing financial interest.

DATAAVA I L A B I L I T YS TAT E M E N T
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

PATI E NTCO N S E NTFO RPU B LI C ATI O N
Informed consent was obtained from all individual participants included in the study.