3.1. STL001 decreased FOXM1 protein expression levels in human cancer cells of different etiology
We have previously described a first-in-class small molecule inhibitor of FOXM1, STL427944, which reduces the chemoresistance of cancer cells by inducing FOXM1 degradation [5]. The STL427944 was identified by transcriptomic network analysis and confirmed in various cancer cell lines as a selective inhibitor of FOXM1 at very high concentrations [5]. However, from a medicinal chemistry perspective, the compound STL427944 has metabolic liabilities,to overcome these issues; we did structural modifications by applying several paths of SAR optimizationand verified the potency, among the newly designed STL427944 analogues, STL001 (Fig. 1A) showed up to a 50-fold estimated increase in potency as FOXM1 inhibitor in AML [39].The novel FOXM1 inhibitor STL001 is a new molecule with similar biological properties to the parent compound STL427944, however the ring replacement in the parental compound is likely to have significantly improved the overall stability and better drug-like properties and thus enhanced potency observed in its derivative, STL001 [39].To assess experimentally the FOXM1-suppressing effect of the STL001 in solid cancer, we used a panel of human solid tumor-derived cell lines with high FOXM1 expression levels, including ovarian cancer (OVCAR-8, ES-2), colorectal cancer (HCT-116, HCT-FET), esophageal cancer (FLO-1),hormone receptor-positive (TAM-R) and triple negative (HCC-1143) breast cancers, and prostate cancer (22RV1, LNCaP). All cell lines were treated with STL001 (1, 5, and 10 µM) resulting in a dose-dependent reduction of the cellular levels of FOXM1 protein at significantly lower concentrations (1 µM, Fig. 1C-G) when compared with its precursor (STL427944) that shows modest FOXM1 suppression at concentrations of 25–50µM (Fig. 1B). These results demonstrate that the STL001 is a universal inhibitor of FOXM1 in cancer cells, also it is 25–50 times more efficient in reducing the cellular FOXM1 activity in solid cancer as compared to its parental compound STL427944 [5].The parental compound, STL427944 affects FOXM1 activity via a two-step mechanism, first, it induces the translocation of nuclear FOXM1 to the cytoplasm, followed by autophagosomaldegradation of FOXM1 protein [5]. We have recently reported that STL001 also induces cytoplasmic re-localization and autophagic degradation of FOXM1 in AML [39]. In the present study, to further verify the induction of autophagy by STL001, we examined the autophagy marker protein, LC3- II/I. Indeed, treatment of C3-luc cells with STL001 for 24 hours resulted in reduced FOXM1 levels with increased expression of autophagy marker protein LC3 (Fig. 1H), the ratio of LC3 II/I was significantly high in C3-luc cells treated with STL001 (Fig. 2I). The same effect was observed in FLO-1 cells treated with STL001 (Fig. 1H and I).Consistent with our recent report in AML [39], these results demonstrate that the first-generation modification drug, STL001,preserves the mode of action of the parentcompound, STL427944, however,it is more efficient in reducing FOXM1 activity in cancer.
3.2. STL001-induced FOXM1 suppression sensitizes human cancers of different origin to a broad-spectrum of cancer therapies.
Chemoresistance is a major barrier for the traditionally used anti-cancer drugs,while FOXM1 overexpression is closely associated with chemoresistance [3, 13, 14, 38] and poor survival in most solid tumors [6, 9]; whereas, FOXM1 down-regulation is proven very effective in restoring chemotherapy sensitivity in several human cancers [5, 38, 39]. Considering FOXM1 as a critical regulator of sensitivity and resistance in human cancers, we assumed that STL001 treatment-induced FOXM1 suppressionshould reduce chemoresistance and sensitize human cancer cells to the cytotoxic effects of the relevant cancer chemotherapies. In the presentstudy, we have explored the sensitization effects of STL001 in combination with a broad spectrum of relevant anticancer drugs with different mechanisms of action: direct DNA damage (cisplatin, doxorubicin, and irinotecan), DNA synthesis inhibition (5-FU), mitosis disruption (paclitaxel), and a selective estrogen receptor modulator (tamoxifen) in a verity of human solid cancer-derived cell lines of different etiology.
In this scenario, initially, we have tested the synergy of STL001 with traditionally used anticancer treatment options in esophageal cancer [44].Esophageal cancer is a highly aggressive malignancy of the gastrointestinal tract with 5-year patient survival ranging from 10 to 20% depending on molecular characteristics. Due to high mutational frequency and high ability of invasion, EC ranks 7th in incidence and the 6th leading cause of cancer-related mortality worldwide [47].Recently, over-expression of FOXM1 has been associated with malignant progression of esophageal cancer [7]. Cisplatin, irinotecan, 5-FU, and paclitaxel are traditionally used in esophageal cancer treatment. However, resistance of esophageal cancer to chemotherapeutic agents, e.g., 5-fluorouracil, cisplatin, and paclitaxel, is a major challenge to successfully treat this malignancy [47].In the present study, esophageal cancer (FLO-1) cells treated with cisplatin at sublethal concentrations significantly increased cellular FOXM1 protein abundance (Fig. 2A). The addition of STL001 in combination with cisplatin efficiently prevented cisplatin-induced FOXM1 activation, resulting in reduced FOXM1 protein levels when compared with corresponding controls (Fig. 2A). Notably, as a single agent, STL001 did not exert significant cytotoxic effects (Fig. 3A), but cells treated with cisplatin chemotherapy in combination with STL001 led to potent induction of apoptotic cell death (indicated by caspase-3 cleavage) when compared with cells treated with cisplatin chemotherapy alone, indicates a strong synergistic apoptotic effect of cisplatin chemotherapy in combination with STL001 (Fig. 2A).
Further, we investigated the synergy between STL001 and one of the best-studied DNA topoisomerase I inhibitors class of drug, irinotecan that prevents religation of the DNA strand and causes double-strand DNA breakage [45]. Similar to the cisplatin effect, the treatment of esophageal cancer cells with irinotecan significantly enhanced FOXM1 protein levels without prominent induction of cell death (Fig. 2C). Combination with STL001 efficiently prevents irinotecan-induced FOXM1 up-regulation and significantly enhances the sensitivity of esophageal cancer cells to the cytotoxic effects of irinotecan therapy (Fig. 2C). Thus, FOXM1 inhibition by STL001 can sensitize esophageal cancer cells to chemotherapies based on DNA damage induction.
While platinum-based agents (cisplatin) and topoisomerase I inhibitors (irinotecan) damage DNA directly, 5-FU treatment interferes with thymidine nucleotide synthesis, which may result in indirect DNA damage [46]. Similar to cisplatin or irinotecan effects, treatment of esophageal cancer cells with 5-FU shows significantly elevated FOXM1 levels without evident cell death induction (Fig. 2E). Combination with STL001 remarkably decreased 5-FU-induced FOXM1 levels and significantly enhanced the sensitivity of esophageal cancer cells to the cytotoxic effects of 5-FU therapy (Fig. 2E). Thus, FOXM1 inhibition by STL001 can sensitize esophageal cancer cells to direct or indirect DNA damage-inducing therapies.
Taxanes (Paclitaxel or Docetaxel) are another class of antitumor drugs, that have been traditionally used in esophageal cancer treatment [45, 47]. However, chemoresistance associated with high FOXM1 levels in cancer cells decreased the efficacy of these therapies against cancer cells [5, 47]. However, unlike DNA-damaging agents (eg, cisplatin, irinotecan, or 5-FU), taxanes are known to modify mitotic spindle microtubule depolymerization dynamics instead of disrupting cell division [48]. In line with our previous results, esophageal cancer cells treated with paclitaxel (Taxol) at sublethal concentrations showed significantly higher levels of cellular FOXM1 protein without showing prominent cytotoxic effects (Fig. 2G). However, STL001 synergizes with taxol to enhance the cytotoxic effects of taxol-chemotherapy, detected by induction of strong apoptotic cell death indicated by caspase-3 cleavage (Fig. 2G). The synergy between STL001 and taxol-chemotherapy indicates that the functional role of FOXM1 as an inducer of drug resistance is not limited to DDR regulation and can be much more universal.
Caspases are the primary drivers of apoptotic cell death and caspase-3 cleavage and activation is a common event in apoptotic cell death. While caspases-3 is well-known to play a central role in apoptosis, we sought to verify cell death using a different method for better reliability. Therefore, Trypan blue dye exclusion assay with direct counting was used to assess the cytotoxic effects of STL001 in combination with other drugs (Fig. 2B, D, F, H). In strong agreement with trends observed using the immunoblotting approach, Figs. 2B, D, F, and H illustrate the significant increase in cell mortality in FLO-1 cells after exposure to STL001 in combination with other drugs for 24 hours. These results confirmed that STL001 sensitizes esophageal cancer cells to cytotoxic effects of a broad spectrum of the relevant esophageal cancer therapies.
Further, we assess whether this compound sensitizes esophageal cancer cells to different chemotherapeutic drugs via mechanisms besides FOXM1 suppression. To test this, we generated FLO-1 cells with stable shRNA-mediated FOXM1-knockdown (FOXM1-KD). As expected, FOXM1-deficient FLO-1 cells showed increased sensitivity to irinotecan (Fig. 2I) and taxol (Fig. 2J), detected by potent induction of apoptosis indicated by caspase-3 cleavage (Fig. 2I and J); however, the sensitization effect of STL001 was absent in FLO-1cells with stable FOXM1-KD (Fig. 2I and J). These findings suggest that FOXM1 is a crucial factor in esophageal cancer chemoresistance and mediates the effects of STL001 in the sensitization of esophageal cancer to different chemotherapies.
In this section of the study, initially, we have verified that the STL001-mediated FOXM1 suppression sensitizes esophageal cancer cells to a broad spectrum of anti-cancer therapies (Fig. 2). Considering thesefindings, we assumed that STL001-induced FOXM1 suppression should reduce chemoresistance in any of the different etiology of cancer cells.In this scenario, finally, we have tested the synergy of anticancer drugs with STL001 in model cell lines belonging to solid tumors (such as ovarian cancer, colorectal cancer, breast cancer, and prostate cancer) of different etiology.
Ovarian cancer is the deadliest disease in women with a very poor 10-year survival rate (< 40%) worldwide. The aberrant over-expression and activation of FOXM1 is the key molecular alteration in ovarian cancers and is associated with poor prognosis and chemotherapy resistance [2]. Doxorubicin is one of the most commonly used anticancer drugs approved by the FDA for ovarian cancer, and it is one of the most important drugs used as a second line of chemotherapy for platinum-resistant patients [49]. Ovarian cancer (OVCAR-8, ES-2) cells treated with sublethal concentrations of doxorubicin, display a significant increase in FOXM1 protein abundance, whereas the addition of STL001 in combination with doxorubicin efficiently prevented FOXM1 activation, resulting in decreased FOXM1 protein levels in comparison with the corresponding control samples (Fig. 3A). Notably, as a single agent, STL001 did not exert significant cytotoxic effects (Fig. 3A), but cells treated with doxorubicin chemotherapy in combination with STL001 led to potent induction of apoptotic cell death (indicated by caspase-3 cleavage) when compared with cells treated with doxorubicin chemotherapy alone, results indicate a strong synergistic apoptotic effect of doxorubicin chemotherapy in combination with STL001 (Fig. 3A). Further, we assess whether STL001 sensitizes ovarian cancer cells to doxorubicin chemotherapy via mechanisms besides FOXM1 suppression. To test this, we used OVCAR-8 cells with stable shRNA-mediated FOXM1-knockdown (FOXM1-KD). As expected, FOXM1-deficient OVCAR-8 cells showed increased sensitivity to doxorubicin (Fig. 3B), detected by potent induction of apoptosis indicated by caspase-3 cleavage (Fig. 1B); however, the sensitization effect of STL001 was absent in OVCAR-8 cells with stable FOXM1-KD (Fig. 3B). These findings suggest that FOXM1 is a crucial factor in ovarian cancer resistance to doxorubicin chemotherapy and mediates the effects of STL001 in the sensitization of ovarian cancer cells.
Further, we study the sensitization effects of STL001 in colorectal cancer, which is among the most lethal and prevalent malignant tumors worldwide. The colorectal cancer meta-analysis associates high FOXM1 expression with a poor 5-year survival rate of colorectal cancer patients [50].5-FU is one of the most frequently used chemotherapy for the treatment of solid cancers. Also, it is the main first-line chemotherapy used for colorectal cancers; however, resistance to 5-FU therapy exists, resulting in a low 5-year survival rate [51, 52].Similar to doxorubicin effects, treatment of colorectal cancer cells (HCT-116 and HCT-FET) with 5-FU resulted in FOXM1 up-regulation without evident cell death of colorectal cancer cells (Fig. 3C). Whereas, the combination of STL001 and 5-FU therapy remarkably decreased 5’FU-induced FOXM1 levels and significantly enhanced the sensitivity of colorectal cancer cells to the cytotoxic effects of 5’FU therapy (Fig. 3C). Moreover, stable shRNA-mediated FOXM1-KD in HCT-116 cells showed increased sensitivity to 5-FU therapy, detected by potent induction of apoptosis indicated by caspase-3 cleavage (Fig. 3D); however, the sensitization effect of STL001 was absent in HCT-116 cells with stable FOXM1-KD (Fig. 3D). Consistent with our findings, FOXM1 has a vital role in 5-FU therapy resistance in colorectal cancer and mediating the synergistic response of STL001 with 5-FU therapy.
Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer death in males.FOXM1 transcription factor is highly expressed in prostate cancer cells and the aberrant over-expression of FOXM1 contributes to prostate cancer development and taxanes resistance [53].Taxanes (paclitaxel or docetaxel) are a different class of chemotherapy drugs that act by binding to tubulins/microtubules and suppressing microtubule dynamics during cell division, paclitaxel and docetaxel are similar in function and widely used to treat a variety of human cancers due to their unique anticancer activity [54]. Clinically, paclitaxel is commonly used as an effective natural antineoplastic drug for the treatment of prostate cancer. However, tumor cells develop resistance to paclitaxel, restricting its application for the treatment of cancer patients [55]. In line with our previous results, prostate cancer (22RV1, LNCaP) cells treated with paclitaxel (Taxol) at sublethal concentrations showed a prominent increase in cellular FOXM1 protein levels without showing any cytotoxic effects (Fig. 3E). However, the treatment of prostate cancer cells with STL001 in combination with taxol enhanced the cytotoxic effects of taxol therapy, detected by induction of strong apoptotic cell death indicated by caspase-3 cleavage (Fig. 3E), these results are consistent with the synergistic effects of STL001 and paclitaxel in esophageal cancer cells (Fig. 2G and H). This data further confirms that FOXM1 is a universal factor involved in therapeutic resistance in cancer cells.
Breast cancer is the most commonly diagnosed cancer in women, about 80% of all breast cancers are positive for estrogen receptors (ER+). Many studies have shown that FOXM1 is highly expressed in different types of breast cancer and its expression was closely associated with poor prognosis and chemotherapy resistance in breast cancer patients [3]. Currently, endocrine therapy is a major treatment option for ER + breast cancer. Tamoxifen is a selective estrogen receptor modulator (SERM), and it is commonly used to treat all stages of hormone-dependent or ER + breast cancers, however, the efficacy of tamoxifen as a breast cancer therapy is not satisfactory because of the development of resistance to tamoxifen [56]. In this context, we determine the role of FOXM1 in tamoxifen resistance, in the present work, tamoxifen treatment of TAMR cells resulted in FOXM1 up-regulation without prominent cell death induction. Whereas, the combination of tamoxifen and STL001 efficiently prevents tamoxifen-induced FOXM1 up-regulation and drastically enhances the cytotoxic effects of tamoxifen therapy, detected by induction of strong apoptosis indicated by caspase-3 cleavage (Fig. 3F) and loss of TAMR cell viability (Fig. 3G). The results have confirmed that the combination of tamoxifen plus STL001 could restore the sensitivity to tamoxifen in tamoxifen therapy-resistant ER + breast cancers.
Besides, it will be of interest to test STL001 in combination treatments to target triple-negative breast cancer (TNBC). TNBC is an aggressive form and accounts for 15–20% of all breast cancers. TNBC cells don’t have estrogen or progesterone receptors and the protein called HER2. However, FOXM1 is highly upregulated in TNBC [57] and has a significant role in the drug resistance of TNBC [3]. Considering this fact, TNBC (HCC-1143) cells were treated with direct DNA-damaging agents (Cisplatin or Doxorubicin) at sublethal concentrations showed significantly higher levels of cellular FOXM1 protein without showing prominent cytotoxic effects (Fig. 3H-I). However, combination with STL001 efficiently prevents cisplatin or doxorubicin chemotherapy-induced FOXM1 up-regulation and drastically enhances the cytotoxic effects of both the DNA-damaging agents, detected by induction of strong apoptotic cell death indicated by caspase-3 cleavage (Fig. 3H-I). These results indicate that STL001 has a strong synergy with DNA-damaging agents (cisplatin or doxorubicin) to induce pro-apoptotic effects in TNBC.
We assess STL001 further to verify if STL001 can sensitize TNBC cells through other mechanisms besides FOXM1 suppression. To study this, we used TNBC (HCC-1143) cells with stable shRNA-mediated FOXM1-KD (Fig. 3J). As expected, HCC-1143 cells with FOXM1-KD show increased sensitivity to doxorubicin, detected by potent induction of apoptosis indicated by caspase-3 cleavage; however, the sensitization effect of STL001 was absent in FOXM1 deficient HCC-1143 cells (Fig. 3J), suggesting that FOXM1 is the main mediator of STL001 effects on TNBC chemoresistance.
In this study, we found that STL001 was effective in sensitizing a wide variety of cancer cells to a broad spectrum of anticancer drugs, through FOXM1 suppression, suggesting that FOXM1 is a crucial factor in therapeutic resistance in solid cancer and mediates the effects of STL001 in sensitization of solid cancer to different chemotherapies.
3.3. RNA-seq analysis of the effects of STL001 and FOXM1-KD on global FOXM1 regulatory network
In the present study, we have examined that STL001 is very effective in sensitizing a wide variety of cancer cells to a broad spectrum of anticancer drugs through FOXM1 suppression. STL001 is a novel small molecule inhibitor of FOXM1, therefore its biological activities and molecular targets need to be characterized. In this perspective, we therefore investigated whether STL001 affects any other pathways besides the FOXM1 target gene network. To achieve that, we did full transcriptome RNA-seq and analyzed the patterns of gene expression by STL001 in esophageal cancer (FLO-1) cells (Fig. 4) and differential gene expression shared between STL001 treatment and FOXM1-KD in ovarian cancer (OVCAR-8) cells (Fig. 5). Processed data on differential gene expression by STL001 or FOXM1-KD are available in supplementary tables 2, 3 and 4).
In this study, out of 16275 protein-coding genes evaluated, we identified a set of 947 genes showing highly significant (2-fold or more) differential expression (DE) in the STL001 treated experimental model, with 757 genes being upregulated and 190 genes being repressed in FLO-1 (Fig. 4A).We therefore considered the genes displaying expression changes in FLO-1 as the most reliable STL001 responders, we pooled them into the “STL001 signature” gene list (947 DE genes), and subjected them to further analysis. We performed Gene Ontology enrichment analysis of the 947 DE genes, in the category of biological process (Fig. 4B). The purpose of this study was to determine whether the DE genes had significant enrichment trends in some functional types. Functional classification found total 12 significantly enriched processes, nine categories were significantly decreased and 3 were significantly increased by STL001 (Fig. 4B). Notably, the mitotic cell division and DNA replication processes were mainly decreased by STL001 (Fig. 4B), suggesting that STL001 mainly affected the FOXM1-targeted gene network. Additionally, cluster analysis was performed on DE genes (947) and 10 significantly enriched biological processes using the GOnet tool. This analysis highlighted clearly linked functional clusters under FOXM1 regulation, such as cell cycle, mitotic cell division, spindle assembly, and DNA replication (Fig. 4C). One cluster related to steroid/cholesterol biosynthetic process and negative regulation of protein secretion was also linked with the core biological functions affected and highlighted with all the upregulated genes (Fig. 4C).
Further, to verify STL001 selectivity toward FOXM1 regulatory pathways, Gene-Set Enrichment Analysis (GSEA) was performed for “STL001 signature” genes (947 DE genes) using canonical pathway gene signatures database, PID. Out of 947 signatures analyzed, 8 gene sets were significantly enriched in FLO-1 cells treated with STL001 (Fig. 4D). Out of 8 gene sets, 7 displayed negative normalized enrichment scores (Fig. 4D), predicting inactivation of these pathways by STL001. It is noteworthy that AURKB and PLK1 are the well-known targets of FOXM1 through direct interaction with their promoters [41], while ATR, BARD1, and FANCONI are DNA damage response (DDR) pathways, several crucial gene components of these pathways are regulated by FOXM1 in DNA damage repair [42]. Moreover, E2F pathway activity can be affected by FOXM1 expression [43], implying that the pathways affected by STL001 converge to the FOXM1-regulated protein network that is involved in tumor survival and resistance to drugs. Taken together, this data shows a very high probability of FOXM1 being the main mediator of STL001 effects on gene expression program in esophageal cancer cells.
To further confirm the selectivity of STL001 toward FOXM1 regulatory pathways we analyzed the differential gene expression shared between STL001 treatment and FOXM1-KD in ovarian cancer (OVCAR-8) cells via full Transcriptome RNA-seq. Figure 5A shows the hierarchical clustering of the differentially expressed genes (DEGs). In Fig. 5B, the differences and overlaps of genes in the STL001 and FOXM1-KD groups are shown using a Venn diagram. Out of 16275 protein-coding genes evaluated, we identified a set of 830 and 2502 genes showing highly significant (2-fold or more) differential expression (DE) in the STL001 and the FOXM1-KD experimental model, respectively (Fig. 5B). In the STL001 group, 535 genes are upregulated and 295 genes are repressed in OVCAR-8 cells, whereas in the FOXM1-KD group, 1497 and 1005 genes are up- and down-regulated, respectively (Fig. 5B). We found that 204 up-regulated DE-genes (~ 62%) and 79 down-regulated DE-genes (~ 37%) by STL001 overlapped with the FOXM1-KD group (Fig. 5B), indicating that STL001 affects the FOXM1 gene network. The Fig. 6C scatter plot of log2 gene expression changes in STL001 treatment and FOXM1-KD shows a correlation between DE genes by STL001 treatment and FOXM1-KD (Spearman’s rho = 0.42). This overlap of transcriptomic effects caused by either STL001 or FOXM1-KD confirms the idea that the effects of STL001 are through suppression of FOXM1 activity.
Further, Gene-Set Enrichment Analysis (GSEA) was performed for “STL001 signature” genes (830 DE genes) and 2502 DE genes in FOXM1-KD using the canonical pathway gene signatures database, PID.In GSEA analysis of DE genes of STL001 or FOXM1-KD group, 3 gene sets were significantly enriched in both thegroups (Fig. 5D). Two gene sets (PID_AURORA_B_Pathway and PID_FOXM1_Pathway) displayed negative normalized enrichment scores (Fig. 5D), predicting the inactivation of these pathways by STL001 or FOXM1-KD. However, only one gene set (PID_NFAT_TFpathway) displayed a positive normalized enrichment score.The FOXM1_Pathway in PID (Fig. 5D) is a predefined collection of the FOXM1 transcription factor network that is involved in cell cycle regulation and DNA damage repair, and it promotes tumor cell proliferation. A total of 40 genes from 7 different gene families are engaged in this pathway, including tumor suppressors, the oncogenes, genes encoding cyclins and cyclin-dependent kinases, different transcription factors, and protein kinases, e.g., such as PLK1 and AURKB, as well as FOXM1 itself. FOXM1 pathway is the top enriched pathway in many human cancers [5, 44]. It is noteworthy that PID_AURORA_B_Pathway (Fig. 5D) which is involved in the proliferation of cancer cells by positive regulation of cell cycle and G2/M phase transition also represents the activity of direct FOXM1 downstream effectors [5, 45]. Moreover, some of the stress response genes involved in the PID_NFAT_TFpathway (Fig. 5D)can be affected by FOXM1 expression, implying that the pathways affected by STL001 or FOXM1-KD converge to the FOXM1-regulated protein network. Moreover, DE genes of cholesterol biosynthetic pathways affected by STL001 in both the FLO-1 and OVCAR-8 exhibited a prominent positive correlation with FOXM1-KD (Table 1). Taken together, this data show a very high probability of FOXM1 being the main mediator of STL001 effects on gene expression program in cancer cells.
Table 1
Differential gene expression of cholesterol biosynthetic pathways shared between STL001 treatments and FOXM1-KD.
SN. | Gene Name | Gene expression changes log2 fold Change |
FLO-1_STL001 | OVCAR-8_STL001 | OVCAR-8 (FOXM1_KD) |
1. | DHCR24 | 1.526395424 | 1.426395424 | 1.726395424 |
2. | MSMO1 | 1.953436883 | 1.866905302 | 1.796905302 |
3. | SC5D | 1.213421737 | 1.313421737 | 3.776874947 |
4. | DHCR7 | 2.277264256 | 1.253611991 | 1.323611991 |
5. | RSAD1/2 | 3.299360696 | 1.053616972 | 4.006732744 |
6. | NPC1L1 | 3.529258581 | 4.675371408 | 6.705199651 |
7. | INSIG1 | 2.275638578 | 2.345350987 | 1.037036562 |
8. | TNFAIP3 | 1.150872916 | 1.069915256 | 2.987135848 |
9. | APOE | 1.344291161 | 1.544291161 | 1.408651275 |
10. | HDAC9 | 1.584113601 | 1.736680718 | 2.347779157 |