NFX1‐123: A potential therapeutic target in cervical cancer

NFX1‐123 is a splice variant isoform of the NFX1 gene. It is highly expressed in cervical cancers caused by HPV, and NFX1‐123 is a protein partner with the HPV oncoprotein E6. Together, NFX1‐123 and E6 affect cellular growth, longevity, and differentiation. The expression status of NFX1‐123 in cancers beyond cervical and head and neck cancers, and its potential as therapeutic target, have not been investigated. TSVdb of TCGA was used to quantify NFX1‐123 expression in 24 cancers compared with normal tissues. The NFX1‐123 protein structure was predicted and then submitted to retrieve suitable drug molecules. The top four compounds, found to bind in silico to NFX1‐123, were tested experimentally to determine their effects on NFX1‐123‐related cellular growth, survival, and migration. 46% of cancers (11 of 24 had significant differences in NFX1‐123 expression, with nine having had greater NFX1‐123 expression, when compared with adjacent normal tissues. Bioinformatics and proteomic predictive analysis modeled the three‐dimensional structure of NFX1‐123, and drug libraries were screened for high‐binding affinity compounds using this modeled structure. Seventeen drugs with binding energies ranging from −1.3 to −10 Kcal/mol were identified. The top four compounds were used to treat HPV‐ and HPV+ cervical cancer cell lines, three of which (Ropitoin, R428 and Ketoconazole) reduced NFX1‐123 protein levels, inhibited cellular growth, survival, and migration, and enhanced the cytotoxicity of Cisplatin. These findings highlight cancers expressing high levels of NFX1‐123, and drugs that target it, may reduce cellular growth, survival, and migration, making NFX1‐123 a potential novel therapeutic target.

While the shorter splice variant isoform of NFX1, NFX1-91, is not (Ref 54 Xu M 2010), NFX1-123 is overexpressed in HPV-associated cancers of the cervix 8,9 and the head and neck. 10 These findings point to the importance of NFX1-123 in cancer cell growth, survival, and differentiation, and it is inferred from its high expression in HPVassociated cancers. The potential for NFX1-123 to be a cancer therapy target, however, has not been investigated. Specifically, the expression levels of NFX1-123 in other non-HPV associated cancers, the predicted three-dimensional structure of NFX1-123, or what compounds could bind to NFX1-123, reducing its amount or functionality in cells, have not been determined.
In this study, we utilized the publicly available Transcript Splice Variant database (TSVdb) of The Cancer Genome Atlas Data (TCGA) to quantify the expression of NFX1-123 in 24 cancers relative to normal tissues, modeled the three-dimensional structure of NFX1-123, identified drug compounds in silico that bound to that protein structure, and tested those compounds to determine their effects on the NFX1-123 protein in cervical cancer cell lines. We found three compounds that affected NFX1-123 also independently reduced cellular growth, survival, and migration, and synergized the cytotoxic effect of Cisplatin when combined.

| NFX1-123 expression analysis in cancer
The specific messenger RNA (mRNA) expression of NFX1-123, as a splice variant isoform of the NFX1 gene, was analyzed using TCGA splice variant database TSVdb 11 as described recently. 10 The TSVdb contains splice variant mRNA expression data for 33 different anatomical sites of cancer tissues. Nine cancers were excluded as they did not have comparative adjacent normal tissue expression for NFX1-123. The list of 24 cancers with data from primary tumors and their respective normal tissues is provided in the table below. Message RNA expression data were downloaded from each type of cancer and its adjacent normal tissue, and the expression data of the NFX1-123 isoform (isoform_uc003zsq) in "primary solid tumor" and "solid tissue normal" was separated to quantify differential expression.

| MTi-OpenScreen
MTi-OpenScreen is a web-server used for virtual screening of small compounds, 16  2.10 | Western immunoblot NFX1-123 protein expression was determined by western immunoblot as described previously. 9 Briefly, cervical cancer cell lines were seeded in six-well or 10 cm plates. Twenty-four hours later, they were treated with Ropitoin (5-20 µM), R428

| Clonogenic assay
To determine the survival and colony formation of cancer cells after drug treatment, clonogenic assays were performed. Cervical cancer cell lines were plated onto six-well plates at densities of 1-3 × 10 3 cells per well.
Cells were fixed and stained using 0.5% crystal violet (Sigma-Aldrich) and imaged using a flat-bed scanner. To quantify colony growth relative to the vehicle treatment control, 0.5% SDS was added to each plate to dissolve stained colonies. The solution from each well was diluted 1:10, and the absorbance was read at 540 nm using a Bio-Tek plate reader. Percent colony growth was calculated as a percentage of the vehicle control colony growth (set to 100%).

| Scratch assay
Scratch assays were performed to determine the wound healing abilities of cervical cancer cells after treatment with Ropitoin, R428, or Ketoconazole. Cervical cancer cell lines were plated onto six-well plates at a density of 2 × 10 5 cells per well. Twenty-four hours later, the plate was scratched in a single, straight line using a 200 µL micropipette tip.

| Determination of 3D structure of NFX1-123 and identification of NFX1-123-binding compounds
The three-dimension structure of the NFX1-123 protein is not known, so the sequence of NFX1-123 was submitted to I-TASSER webserver to predict its protein structure. Five model structures were generated, and these structures were submitted to the MTi-Openscreen Virtual screening webserver to retrieve suitable, commercially available drug compounds from the Drug-lib database. Among the five models of NFX1-123, the structure of Model 2 (Figure 2A)  scores that were more than 10 Kcal/mol. (Figure 2B).
Of the 17 drug compounds noted above, the top 10 drugs modeled to bind NFX1-123 were further analyzed to identify their binding sites on NFX1-123, using the autodock docking tool kit followed by the pymol visualization tool, shown in Figure 3B. The

| DISCUSSION
The importance of NFX1-123, in cooperation with 16E6, on cellular growth, immortalization, survival, and differentiation has been shown, 4,5,7,8,19,20 and NFX1-123 is highly expression in cervical cancer cell lines 19 and primary tumors, and in HPV positive head and neck cancers. 9,10 NFX1-123 itself contains a PHD/RING domain, which functions as an E3 ubiquitin ligase, and the deubiquitinase USP9X, which protects the proteins from proteasome-mediated degradation, binds to NFX1-123. 4 Greater expression of USP9X is seen in several tumor types, 21 and 16E6 increases USP9X expression, thus indirectly affecting NFX1-123 levels as well. 21 All these studies point to high expression of NFX1-123 being important in tumor cell growth and survival, specifically in HPV-associated cancers.
There are six known types of cancer that are caused by HPV: cervical, vulvar, vaginal, anal, penile, and head and neck cancers.
NFX1-123 appears to play a foundational and functional role in the cellular dysregulation required by HPV at the very least in cervical cancer. [7][8][9]19 The role NFX1-123 has in cancers more broadly, whether HPV-associated or not, has not been well studied. Using the TSVdb. we found that 46% (11 out of 24) of cancers had altered NFX1-123 expression, 9 of which were increased ( Figure 1A , or a control. Cells were allowed to migrate through the membrane into chemotactic lower chamber for 24 h. The percent cells migrated through the membrane was quantified and normalized to vehicle-treated control. Images were captured at ×10 using the REVOLVE-Discover ECHO microscope. Percent migrated cells were significantly reduced in Rop, R428, and KTZ treated cells compared with control. *p < 0.01, **p < 0.001, *** p < 0.0005.
protein targets and perform virtual screens to identify compounds which bind a target, is gaining prominence because it is inexpensive and fast. [23][24][25][26] In this study, we leveraged this in silico work to identify drug compounds that may bind to, and affect, NFX1-123.
First, the predicted three-dimensional structure of NFX1-123 was determined ( Figure 2A) and then utilized as a model to screen ligand drug compounds. A total of 894 were identified whose three-dimensional structures were available in ZINC database and which had strong drug: Ropitoin (TR 2985) is an antiarrhythmic drug that depresses the maximum upstroke velocity of the action potential in a voltagedependent fashion. 29 While the voltage-gated sodium and potassium channels have been shown as potential therapeutic targets and biomarkers in cancers, [30][31][32] and specifically in cervical cancer, [33][34][35] the direct effect of Ropitoin on sodium or potassium channels, let alone NFX1-123, has not been investigated. Our findings make Ropitoin an interesting drug for further study in cancers and as a voltage-gated channel regulator.
R428 (Bencentinib) is an inhibitor of Axl, the receptor tyrosine kinase. It is currently under evaluation as a drug therapy in several cancer clinical trials. 36 Axl has been demonstrated to be a therapeutic target in head and neck cancers 37 and cervical cancers, 38 and Axl has a role in cancer therapeutic resistance. [39][40][41] Axl is a known target of R428, but R428 also affects several biological processes including apoptosis, lysosomal acidification and recycling, and the immune microenvironment. Because R428 has already been shown to affect HPV-associated cancers, [37][38][39] our data adds to those findings.
Ketoconazole (KTZ) is a FDA-approved antifungal drug. Interestingly, studies have identified Ketoconazole's anticancer activities, 42,43 and clinical trials are ongoing to test its efficacy against cancer (NCT00895310, NCT03796273). Ketoconazole inhibits adrenal testosterone synthesis through the inhibition of cytochrome P450 14α-demethylase. 44 This implies that Ketoconazole could function in the treatment of metastatic, castration-resistant prostate cancer, leading to improved survival. 45 In renal cancer, which has increased expression of NFX1-123 (Figure 1), Ketoconazole has been shown to be an inhibitor of exosome production, and as an adjunct therapy, Ketoconazole potentiates the effectiveness of Sunitinib by decreasing tumor cell proliferation, and the clonogenic potential of renal cancer cell lines. 46 All three of these drug compounds decreased NFX1-123 protein in cervical cancer cell lines and were associated, in a dose-dependent manner, with the downregulation of key indicators of cellular growth and metastatic potential; however, the final drug compound modeled to bind to NFX1-123, Perospirone Hydrochloride, did not reduce NFX1-123 levels. This emphasizes the importance of validation studies, and that most, but not all, of these drug compounds did appear to target the NFX1-123 protein, and its effects on cellular growth, in cervical cancer cell lines.
In conclusion, we interrogated publicly available databases to identify nine cancers, beyond HPV-associated cervical and head and neck cancers, that had higher expression of NFX1-123 relative to their normal tissues. Through in silico modeling, we also found three drug compounds that were predicted to bind to NFX1-123. These drug compounds reduced NFX1-123 protein and inhibited cervical cancer cell line growth, survival, and migration, singularly or in combination with Cisplatin. These findings suggest that NFX1-123 may be a potential therapeutic target in cancer treatment.