PBK/TOPK Is a Favorable Prognostic Biomarker Correlated with Antitumor Immunity in Colon Cancers

Immune checkpoint inhibitor therapy has proven efficacy in a subset of colon cancer patients featuring a deficient DNA mismatch repair system or a high microsatellite instability profile. However, there is high demand for more effective biomarkers to expand the colon cancer population responding to ICI therapy. PBK/TOPK, a serine/threonine kinase, plays a role in cell cycle regulation and mitotic progression. Here, we investigated the correlation between PBK/TOPK expression and tumor immunity and its prognostic value in colon cancer. Based on large-scale bioinformatics analysis, we discovered that elevated PBK/TOPK expression predicted a favorable outcome in patients with colon cancer and was positively associated with immune infiltration levels of CD8+ T cells, CD4+ T cells, natural killer cells, and M1 macrophages. In contrast, a negative correlation was found between PBK/TOPK expression and immune suppressor cells, including regulatory T cells and M2 macrophages. Furthermore, the expression of PBK/TOPK was correlated with the expression of T-cell cytotoxicity genes in colon cancer. Additionally, high PBK/TOPK expression was associated with mutations in DNA damage repair genes, and thus with increased tumor mutation and neoantigen burden. These findings suggest that PBK/TOPK may serve as a prognostic and predictive biomarker for immunotherapy in colon cancer.


Introduction
Colon cancer is a malignant disease ranked third in cancer incidence and second in cancer mortality worldwide [1]. The incidence and mortality have been slowly declining each year, mainly owing to the surgical resection of primary tumors at the early localized stages. The 5-year survival rate at a late stage, however, remains profoundly low [1]. Targeted therapy, such as cetuximab or bevacizumab, has been shown to prolong overall survival (OS), but it is effective only in a subset of patients with colon cancer [2]. Two immune checkpoint inhibitors (ICIs) that target programmed cell death-1 (PD-1) have been FDA-approved for the treatment of patients with colon cancer exhibiting high microsatellite instability (MSI-H) or DNA mismatch repair (MMR) deficiency (dMMR) [3,4]. However, response to anti-PD-1 immunotherapy is highly variable, and tumor mutational burden (TMB) alone is insufficient to predict responses in colon cancers [5]. Therefore, the development of effective predictors of immunotherapeutic response for patients with colon cancer is crucial.

Materials and Methods
Gene expression and clinicopathological data of colon cancer patients and healthy volunteers were collected and obtained from public databases, as described below.

PBK/TOPK gene expression analyses of tumor and normal samples in The Cancer
Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) were performed on the GEPIA2 web portal (http://gepia2.cancer-pku.cn/, accessed on 13 October 2021) [28]. The gene expression data of 1372 Cancer Cell Line Encyclopedia (CCLE) cell lines were obtained from the Dependency Map (DepMap) Public 21Q3 dataset on the DepMap web portal (https://depmap.org/portal/, accessed on 15 September 2021). Normal tissue gene expression data were obtained from the GTEx portal (https://gtexportal.org/, accessed on 8 August 2021). PBK/TOPK mRNA expression of the CCLE and GTEx was visualized using the programming software R (version 4.1.1, https://www.r-project.org/, accessed on 8 August 2021). To confirm the expression profile of TCGA and GTEx, other gene expression data for colon tumor samples and paired normal samples were downloaded from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/, accessed on 15 September 2021): GSE44076. In total, 98 pairs of tumor and normal tissue data were analyzed for differential PBK/TOPK expression. To analyze methylated CpG sites of the PBK/TOPK promoter, Human Methylation 450K data were downloaded from the UCSC Xena browser (https://xenabrowser.net/, accessed on 10 September 2020) [29]. Immunohistochemical images of PBK/TOPK protein expression were obtained from the Human Protein Atlas (http://www.proteinatlas.org, accessed on 14 October 2021).

Survival Analysis
Survival analysis between PBK/TOPK-high and -low groups was performed with the GEPIA2 web portal across TCGA cancer types [28]. Briefly, TCGA patient samples were sub-grouped into two cohorts according to the 25% cutoff expression values. The OS and disease-free survival (DFS) of the two cohorts were visualized using Kaplan-Meier curves with hazard ratios (HRs) of the Cox-PH model and log-rank p values. A meta-analysis was performed to assess the correlation between PBK/TOPK gene expression and prognosis across a variety of solid tumors. Prognosis data were downloaded from the PROGgeneV2 database (http://www.progtools.net/gene/, accessed on 27 July 2019) [30] and cohorts with sample numbers over 60 were included. HRs with 95% CI and log-rank p values across 54 cancer cohorts were displayed with a forest plot.

Differential Gene Expression and Correlation Analysis
RNA-sequencing gene expression and mutation data of TCGA and CPTAC-2 were downloaded from the GDC Data Portal (https://portal.gdc.cancer.gov/, accessed on 23 September 2021) using TCGAbiolinks R/Bioconductor package or the cBioPortal webpage [38,39]. For the differential expression (DE) gene analysis, TCGA or CPTAC-2 cohorts were divided into two subgroups according to the PBK/TOPK expression levels ("PBK/TOPK-high" and "PBK/TOPK-low" represented the cut-off values of the top and bottom 10% levels, respectively). A DE gene analysis between the two groups was performed using the limma R/Bioconductor package [40]. To investigate the pathway, a gene set enrichment analysis (GSEA) was conducted with MSigDB (version 7.4) using the fgsea R/Bioconductor package [41]. A pre-ranked gene matrix for GSEA was calculated using the limma t-statistic or Pearson's correlation coefficient with PBK/TOPK.

Immunoblotting
The antibody against PBK/TOPK was purchased from BD Biosciences (San Jose, CA, USA). Anti-GAPDH antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Cells were lysed using an M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1× Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated on an SDS-polyacrylamide gel and transferred onto a PVDF membrane (MilliporeSigma, Burlington, MA, USA). The signals were developed using an enhanced chemiluminescence detection system (ElpisBio, South Korea) and visualized with Chemi-Doc (Bio-Rad Laboratories, Hercules, CA, USA).

Cell Cycle Analysis
Wild-type (WT) and PBK/TOPK-KO HCT-116 cells were incubated for one day in a humidified incubator with 5% CO 2 after seeding. To induce DNA damage, HCT-116 cells were treated with SN-38 (Selleckchem, Houston, TX, USA) or with vehicle at the indicated concentrations and time points. Cells were fixed in ice-cold 70% ethanol and stained with propidium iodide solution (BioLegend, San Diego, CA, USA). The cell cycle was analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA).

Statistical Analysis
Statistical analysis was performed using the GraphPad Prism (version 9.2.0, La Jolla, CA, USA) software and R (version 4.1.1, https://www.r-project.org/, accessed on 8 August 2021). Significance was determined using the two-tailed paired t-test for comparing tumor and matched normal samples. Student's t-test was used for the independent two-group comparison, one-way ANOVA was used for the multiple group comparison, and the logrank test was used for the survival analysis. p values < 0.05 were considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

High PBK/TOPK Expression Shows a Good Prognosis in Colon Cancer
PBK/TOPK is known to be highly expressed on various malignant cells compared to normal tissues, which show a limited level of PBK/TOPK expression, except for the testes (Supplementary Figures S1 and S2) [6]. Analysis of TCGA data base revealed that the PBK promoter was hypo-methylated in colon cancer tissues compared with normal tissues, indicating that PBK/TOPK can be epigenetically induced in colon cancers (Supplementary Figure S3). We next examined the prognostic value of PBK/TOPK in colon adenocarcinoma (COAD), breast invasive carcinoma (BRCA), bladder urothelial carcinoma (BLCA), esophageal carcinoma (ESCA), glioblastoma multiforme, lung adenocarcinoma (LUAD), ovarian serous cystadenocarcinoma (OV), and stomach adenocarcinoma (STAD) of TCGA cohort where its tumorigenic roles are proposed [16,[42][43][44][45][46][47][48].  Figure S4). Our metaanalysis of survival data further confirmed a positive correlation between PBK/TOPK expression and OS in colon cancers, but not in ovarian cancers ( Figure 1B). We could not observe a correlation between PBK/TOPK expression and OS in ESCA and STAD owing to a lack of cohorts in the database. We next examined whether PBK/TOPK expression can differentiate patients with colon cancer exhibiting a different prognosis status. Since colon cancer progresses with a stepwise accumulation of genetic or epigenetic alterations and changes [49,50], we assessed PBK/TOPK expression levels in different stages of colon cancer ( Figure 1C). PBK/TOPK expression gradually decreased as colon cancer progressed.
The OS analysis results also demonstrated a correlation between a good prognosis and PBK/TOPK expression in stage I-III colon cancers ( Figure 1D). Together with previous findings [20,21], our analysis supports that PBK/TOPK expression exhibits a favorable prognosis in patients with colon cancer.

PBK/TOPK Gene Expression Is Correlated with Increased Accumulation of Antitumor Immune Cells
Immune cell infiltration is an important parameter for assessing tumor-immune interactions and the consequential effect on OS in colon cancer [51]. Hence, we examined whether a favorable prognosis of PBK/TOPK-high colon cancer was associated with increased accumulation of antitumor immune cells in the tumor microenvironment (TME). We analyzed the cellular composition of the intratumoral immune infiltrates with bulk RNA-sequencing data of TCGA and CPTAC-2 colon cancer by using different deconvolution algorithms in the TIMER2.0 web portal. The result showed a positive correlation between PBK/TOPK expression and the infiltration level of antitumor immune cells, including CD8+ T cells, natural killer (NK) cells, CD4+ T cells, and M1 macrophages. In addition, immunosuppressive protumor immune cells, such as regulatory T (Treg) cells and M2 macrophages, exhibited limited infiltration in PBK/TOPK-high patients with colon cancer (Figure 2A,B). The results were consistent over different deconvolution algorithms. We observed a similar correlation with PBK/TOPK expression when analyzing CD8+ T cell (R = 0.14, p = 0.0074), NK cell (R = 0.014, p = 0.79), and M1 macrophage scores (R = 0.12, p = 0.017) provided by Liu

Association between PBK/TOPK Expression and Antitumor Function of Tumor Infiltrating Immune Cells in Colon Cancers
To understand the functional characteristics of infiltrated immune cells in PBK/TOPKhigh colon cancer, we performed a DE gene analysis between PBK/TOPK-high and -low groups. In total, 1802 (938 up-and 864 downregulated) and 3776 (1222 up-and 2554 downregulated) DE genes were analyzed in TCGA and CPTAC-2 colon cancer samples, respectively. First, we identified cytotoxic T cell markers (IFNG, GNLY, and GZM gene family), T-cell co-stimulatory molecules (ICOSLG and TNFSF9), T cell activation signaling genes (LCK, TBX21, EOMES, MTHFD2, and ISG15), T-cell migration genes (CXCL gene family), and a component of major histocompatibility complex (MHC) class I (B2M) in upregulated DE genes ( Figure 3A). These findings suggest that high PBK/TOPK expression is positively correlated with cytotoxic and inflammatory immune signatures in colon cancer. On the contrary, T-cell inhibitory molecules (PVRIG and CEACAM gene family) and immunosuppressive Treg cell markers (FOXP3) were identified in downregulated DE genes ( Figure 3A). We observed the downregulation of M2 macrophage markers (CD163, MSR1, MAF, and MRC1) but did not reach the DE gene criteria (absolute log2 fold change >1 and adjusted p value < 0.05). We confirmed that the DE gene analysis result was highly consistent with our immune cell infiltration analysis, as shown in Figure 2A. We next investigated the correlation between the expression of PBK/TOPK and genes related to the effector T cell, exhausted T cell, T-cell migration, and antigen presentation. Figure 3B shows that PBK/TOPK expression is significantly associated with the genes expressed in cytotoxic T cells, but not in the exhausted T cells. Furthermore, a positive correlation between the expression of effector T-cell-attracting chemokines (CCL5 and CXCL9) and PBK/TOPK was observed. We also identified that the analyzed DE genes shown in Figure 3A were enriched in pathways related to "effector CD8 T cell up", "naïve CD8 T cell down", and "IFNgamma response up" with statistical significance (p value < 0.05) ( Figure 3C). PBK/TOPK expression had significant positive correlations with the GZMA/CD8A (R = 0.15, p = 0.00081), GZMB/CD8A (R = 0.13, p = 0.0024), and IFNG/CD8A (R = 0.25, p = 8.6 × 10 −9 ) ratio, suggesting that higher expression of PBK/TOPK is positively correlated with increased cytotoxic activity of CD8+ T cells ( Figure 3D). Since PBK/TOPK expression was correlated with not only the degree of CD8+ cell infiltration but also their cytotoxic functions, we considered whether PBK/TOPK expression could represent an antitumor response by CD8+ T cells. We re-analyzed the prognostic value of PBK/TOPK in TCGA COAD cohort. First, we divided the entire cohort into two sub-cohorts: CD8-high and -low, according to CD8A and CD8B gene expression. We then examined the prognostic value of PBK/TOPK in each sub-cohort. In the CD8-low sub-cohort, patients with high PBK/TOPK expression did not show an improved prognosis compared with those with low PBK/TOPK expression. In the CD8-high sub-cohort, on the contrary, high PBK/TOPK expression was associated with a survival benefit in patients with colon cancer (Figure 3E), although the result did not reach statistical significance owing to the small number of patient samples. We next investigated the correlation between PBK/TOPK expression and immune cell infiltration level by tumor stage. The analysis illustrated that a good prognosis of PBK/TOPK was consistent with the degree of immune cell infiltration in stage I-III patients (Figures 1D and 3F). Additionally, the correlation between PBK/TOPK expression and CD8+ T cell infiltration tended to decrease with increasing disease stage, suggesting that CD8+ T cells might be the major immune subtype determining the prognostic value of PBK/TOPK ( Figure 3F) [53]. However, a favorable prognosis of PBK/TOPK in stage IV patients was not observed (Figures 1D and 3F), likely because there exists a more important prognosis-determining factor than immune cell infiltration.

Positive Correlation between High PBK/TOPK Expression and Tumor Mutation Burden
Recently, Liu et al. suggested five molecular subtypes of adenocarcinomas of the GI tract: chromosomal instability (CIN), Epstein-Barr virus-positive, genome stable (GS), hypermutated-single-nucleotide variant (SNV) predominant (HM-SNV), and MSI [54]. Since both MSI and HM-SNV tumors are characterized by high T-cell infiltration and mutation burden in COAD, we sub-analyzed the degrees of CD8+ T cell, NK cell, and M1 macrophage infiltration in COAD. HM-SNV and MSI subtypes exhibited overall increases in the CD8+ T cell, NK cell, and M1 macrophage scores compared with the CIN subtype ( Figure 4A). We next analyzed the PBK/TOPK expression level across four different tumor types of COAD. PBK/TOPK expression was significantly higher in HM-SNV and MSI types compared with the CIN type ( Figure 4B). We confirmed the result with another MSI classification guideline by the Bethesda panel, indicating that MSI-H is associated with high PBK/TOPK expression compared with microsatellite stable (MSS) and MSI-low in TCGA COAD ( Figure 4C). We obtained a similar result in the CPTAC-2 cohort (Supplementary Figure S6A). We conducted GSEA to confirm the correlation between PBK/TOPK expression and MSI status. As shown in Figure 4D, PBK/TOPK co-expressed genes were enriched in the gene set that represents a colon cancer MSI signature. Both HM-SNV and MSI showed hypermutated phenotypes due to defects in DNA repair pathways. We investigated whether PBK/TOPK is related to TMB in TCGA and CPTAC-2 COAD. PBK/TOPK expression was strongly correlated with the total mutation count (TCGA R = 0.42, p = 9.1 × 10 −13 ; CPTAC-2 R = 0.45, p = 1.6 × 10 −6 ) ( Figure 4E and Supplementary Figure S6B). Similar to the total mutation count, the predicted neoantigen count also exhibited a strong positive correlation (R = 0.41, p = 2.9 × 10 −12 ) with PBK/TOPK expression ( Figure 4F). We next examined the association of the PBK/TOPK expression level with KRAS or BRAF mutations. These mutations are known to be crucial in colon cancer pathogenesis. MSI colon cancers with epigenetic silencing of the MLH1 gene are correlated with the BRAF (V600E) mutation, while the KRAS hotspot mutation is greatly associated with MSS tumors and a subset of MSI tumors without defects in MLH1 or MSH2 genes [54]. PBK/TOPK showed a significant correlation only with the BRAF (V600E) but not KRAS (G12/G13) mutation ( Figure 4G). These results reinforce our findings demonstrating the correlation between PBK/TOPK and MSI colon cancers.

PBK/TOPK Is Associated with DNA Repair Pathways
We next conducted a meta-analysis of gene expression array databases with the CO-Regulation Database tool (http://cord-db.org/, accessed on 31 July 2019) [55]. As shown in Figure 5A, PBK/TOPK was significantly concordant with several mitosisrelated pathways, such as "cell cycle", "DNA replication", "pyrimidine metabolism", and "purine metabolism", in-line with the findings of previous studies [12,43]. Pathways related to DNA repair were also identified, including "mismatch repair", "homologous recombination", "nucleotide excision repair", and "base excision repair". We confirmed the results with GSEA. Genes that were co-expressed with PBK/TOPK were enriched in several DNA repair pathways, including the "hallmark DNA repair" gene set of MSigDB ( Figure 5B). The analysis suggested that PBK/TOPK may play a role in the DNA repair mechanism.
In addition, we found that the "Reactome G1/S DNA damage checkpoint" gene set was co-expressed with PBK/TOPK ( Figure 5D). Since PBK/TOPK expression shows high correlations with TMB and DNA repair pathways, we investigated whether the upregulation of PBK/TOPK could enable the cells with DNA damage to bypass cell cycle checkpoints, leading to the accumulation of DNA mutations. We generated PBK/TOPK-KO HCT-116 colon cancer cell lines (Supplementary Figure S7). Knockouts of PBK/TOPK did not affect the rate of cell proliferation under normal culture conditions. However, a significant arrest in the G1 phase was observed in PBK/TOPK-KO HCT-116 cells upon treatment with the DNA-damaging agent SN-38 (an active metabolite of irinotecan) ( Figure 5E), suggesting a possible role of PBK/TOPK at the G1/S checkpoint in response to DNA damage.

Discussion
Although anti-PD-1/PD-L1 antibodies may be effective in treating cancer, their response rates are only approximately 10% to 20% in unselected patients [58]. These response rates could be increased with patient selection. In metastatic CRC patients treated with pembrolizumab (anti-PD-1 antibody), patients with MMR-deficient tumors had a 40% response rate, compared to 0% for those with MMR-proficient tumors. However, only approximately 2-4% of patients with metastatic CRC have dMMR/MSI-high disease and some of them do not respond to anti-PD-1 therapy [59]. Therefore, it is important to discover more effective predictive biomarkers to improve treatment outcomes and extend the benefit of immunotherapy to a greater population of patients with CRC.
In this study, we conducted a large-scale bioinformatic analysis of the genetic and clinicopathologic data of patients with colon cancer and revealed that PBK/TOPK could act as a favorable prognostic biomarker in colon cancer. Given the oncogenic role of PBK/TOPK in the process of tumorigenesis and tumor progression, it is interesting to find that high expression of PBK/TOPK correlates with an improved prognosis in colon cancer. Since tumor infiltrating immune cells contributes to determining the prognosis of colon cancer [60,61], we analyzed the changes in immune profiles including immune cell composition and inflammatory signatures. High PBK/TOPK expression in colon cancer was correlated with an increased infiltration level of cytotoxic immune cells, whereas activated CD8+ T-and NK-cell-infiltrated tumors also exhibited decreased infiltration of Treg cells and M2 macrophages that have immunosuppressive roles in TME. High cytotoxic activity, CD8/Treg ratios, and expression of IFN-γ and granzymes by CD8+ T cells were observed in PBK/TOPK-high colon cancer. Using genomic data drawn from TCGA and CPTAC-2, we found that high PBK/TOPK expression is associated with impaired DNA repair. Mutations in DNA repair genes were enriched in PBK/TOPK-high colon cancers. It has long been known that mutations in DNA repair pathways are associated with higher TMB and neoantigen load [62]. We found that colon cancer patients with high PBK/TOPK expression showed higher MSI and TMB, which is associated with neoantigen burden. Therefore, PBK/TOPK expression levels could serve as a surrogate biomarker for identifying colon cancer patients with high TMB. Further studies are required to determine if analyzing the expression level of PBK/TOPK in colon cancer may be a clinically viable method for predicting TMB levels. Owing to their increased tumor mutational burden, PBK/TOPKhigh colon cancers could present neoantigens on MHC-I molecules and promote high levels of neoantigen specific T-cell activation, ultimately leading to the destruction of cancer cells ( Figure 6). Although PBK/TOPK is also overexpressed in most tumors, we did not observe a favorable prognosis in patients with other types of tumors except COAD. Previous studies have reported that high PBK expression is associated with a poor prognosis in patients with several types of malignancies, such as kidney renal clear cell carcinoma (KIRC), lower grade glioma (LGG), and liver hepatocellular carcinoma (LIHC) [63]. To address this discrepancy, we analyzed the correlation between PBK/TOPK expression and the degree of immune cell infiltration in non-small cell lung cancer (NSCLC). In two types of NSCLC, LUAD and lung squamous cell carcinoma (LUSC), the analysis exhibited a positive correlation of PBK/TOPK expression and a certain degree of immune cell infiltration (Supplementary Figure S8A,B). These observations are similar to those of colon cancer. However, a high PBK/TOPK expression was correlated with a poor prognosis in LUAD ( Figure 1A and Supplementary Figure S4) and a good prognosis in LUSC (Supplementary Figure S8C,D). Another report demonstrated that PBK/TOPK-high esophageal squamous cell carcinoma showed a good prognosis; however, no immune cell infiltration was observed [23]. To understand these differences between cancer types, a comprehensive analysis is needed across all types of cancers with multiple prognostic factors, such as oncogene and tumor suppressor gene mutations, lineage-dependent gene expression, and specific immune cell subtypes. PBK/TOPK appears to promote mitosis for cancer cell proliferation according to our pathway analysis data (data not shown) and those of previous reports [6]; however, how much or how independently PBK/TOPK contributes to the proliferation of colon cancer cells is not clear. A CRISPRbased KO study showed that the expression of PBK/TOPK is not critical for cancer cell proliferation [64]. The dependency scores in the DepMap database also demonstrated that PBK/TOPK may not affect the proliferation of colon cancer cell lines (data not shown). In addition, PBK/TOPK-KO mice do not exhibit any differences in body weight or male reproductive systems [65]. Although these findings provide some support as to why PBK/TOPK upregulation is not associated with a significantly poor prognosis in most cancer types, further studies should be performed to unveil the exact roles of PBK/TOPK under different circumstances. Given that this study has a limited patient cohort size, additional data from prospective studies are needed.

Conclusions
A variety of biomarker strategies for ICI therapy have been extensively explored to identify patient populations that will respond to treatment [66]. In this study, we proposed the association between high PBK/TOPK expression and favorable prognose in patients with colon cancer. Given the correlation among MSI-H, the mutational load, and cytotoxic immune cell infiltration, upregulation of PBK/TOPK in colon cancer could be a potential candidate marker to guide patient selection for immunotherapy.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/biomedicines10020299/s1, Figure S1: PBK/TOPK gene expression in various primary tumors, cell lines, and normal tissues, Figure S2: PBK/TOPK protein expression in colon cancer patient tissue samples, Figure S3: The methylation status of PBK promoter region in TCGA COAD and matched normal samples, Figure S4: Prognostic values of PBK/TOPK in various cancer types, Figure S5: Correlation between PBK/TOPK expression and immune cell infiltration in colon cancer, Figure S6: Correlation between PBK/TOPK expression and MSI subtype in the CPTAC-2 colon cancer samples, Figure S7: PBK/TOPK gene knockout in the HCT-116 colon cancer cell line, Figure S8

Institutional Review Board Statement:
The study was based on the analysis of publicly available datasets and did not need any ethics committee's approval.
Informed Consent Statement: Not applicable. All data used in this study are publicly available and do not violate any rights of people or institutions.
Data Availability Statement: All the data are available online with common access.

Conflicts of Interest:
The authors declare no conflict of interest.