Telmisartan Facilitates the Anticancer Effects of CARP-1 Functional Mimetic and Sorafenib in Rociletinib Resistant Non-small Cell Lung Cancer

Background/Aim: Tyrosine kinase inhibitors (TKIs) are used for the treatment of both wild type and mutant non-small cell lung cancer (NSCLC); however, acquired resistance is a major clinical challenge. Herein, we aimed to investigate the effects of telmisartan (Tel), CFM 4.16 and sorafenib combination in rociletinib resistant NSCLC tumors. Materials and Methods: 3D spheroid cultures and western blotting were used for evaluating cytotoxic effects and protein expression. An in vivo rociletinib resistant H1975 xenograft model of NSCLC was developed by subcutaneous injection of rociletinib resistant H1975 cells into nude mice. Results: Tel, CFM 4.16 and sorafenib combination displayed superior anti-cancer effects in 3D spheroid cultures and a rociletinib resistant H1975 xenograft model of NSCLC by decreasing the protein expression of oncogenic and cancer stem cell markers (Nanog, Sox2 and Oct4). Conclusion: Tel facilitates effective penetration of CFM 4.16 and sorafenib in rociletinib resistant H1975 models of NSCLC.

Lung cancer is the leading cause of cancer-related deaths worldwide for both men and women (1). 50% of the lung cancer patients die within one year of diagnosis and 5-year survival rate is less than 18% (2). Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancers and around 70% of the lung cancer patients are in advanced metastatic stage at the time of diagnosis (3). Multiple studies have demonstrated superior efficacy with first generation (gefitinib and erlotinib), second generation (afatinib) and third generation (osimertinib and rociletinib) tyrosine kinase inhibitors (TKIs) in advanced metastatic lung cancer tumors harbouring epidermal growth factor receptor (EGFR) mutations when compared to standard chemotherapy of cisplatin, paclitaxel and gemcitabine (4)(5)(6)(7)(8).
Rociletinib, an irreversible third-generation tyrosine kinase inhibitor (TKI) is demonstrated to be effective against EGFR T790M mutations in NSCLC through covalent binding with cysteine (C) 797 in the ATP binding pocket of EGFR kinase domain and strong hydrophobic interactions with the gatekeeper methionine (M) residue in T790M (9). However, preclinical trials have shown minimal efficacy of rociletinib against EGFR wild type NSCLC (10). It has been well demonstrated to show better therapeutic effects than first generation TKIs (i.e., erlotinib or gefitinib) for NSCLC harbouring T790M and L858R mutations and brain metastasis (11,12). However, the majority of NSCLC patients develop resistance to rociletinib due to HER2 amplification, MET amplification, BRAF mutation, epithelial-mesenchymal transition (EMT), over-expression of basal phosphorylated AKT levels, ERK activation, EGFR mutations involving substitution of cysteine at position 797 with serine (C797S), lysine at position 844 with valine (K844V), and leucine at position 718 with glutamine (L718Q) (13)(14)(15)(16).
Sorafenib, approved by the FDA for kidney and liver cancers, is a multi-kinase inhibitor of serine/threonine kinases CRAF, BRAF (wild type), as well as the mutant BRAFV600E type (17)(18)(19). A growing body of evidence demonstrates that sorafenib inhibits the activation of VEGR, PDGFR, FGFR, c-KIT, MET, MAPK, and angiogenesis (17,18,20). Sorafenib induces apoptosis and inhibits MEK and ERK phosphorylation in a variety of cancers harbouring BRAF and/or KRAS or NRAS mutations (18,21). Sorafenib, in combination with various molecules targeting EGFR and MET, have shown good anti-cancer activity in wild type and drug resistant lung cancers (19,22).
Cell cycle and apoptosis regulator protein 1/CCAR1 (CARP-1) is a peri-nuclear phosphoprotein that regulates the cell cycle and tumor progression by co-activating the anaphase-promoting complex (APC/C), an E3 ubiquitin ligase (23,24). It also regulates chemotherapy-induced apoptosis through p53 co-activation (25). CARP-1 functional mimetics (CFMs) induce apoptosis and inhibit cell growth in a variety of cancer cells by decreasing the binding of CARP-1 with APC/C subunit APC2 (26). CFM 4.16, a CARP-1 functional mimetic in combination with sorafenib has shown good anticancer activity in TNBC, NSCLC, and renal cancers. This combination also showed excellent anti-cancer efficacy in rociletinib resistant H1975 NSCLC xenograft model (22).
Clinical efficacy of various anti-cancer drugs is limited because of poor diffusion and penetration of drugs into solid tumors owing to high interstitial pressure or tumor stromal barriers (i.e., collagen rich network embedment in hyaluronan of tumor stroma) (27)(28)(29). The role of telmisartan in decreasing tumor interstitial fibrosis and promoting intratumoral distribution of nanoparticles and liposomes is well documented (30)(31)(32). Telmisartan also shows anti-cancer effects in NSCLC by inhibition of PI3K signalling and activation of peroxisome proliferator activated receptor-γ (PPARγ) pathways (33,34).

Materials and Methods
Cell culture. Non-small cell lung cancer cell lines: HCC827, HCC827-R (T790M EGFR ERL Resistant) and H1975 (T790M EGFR ERL Resistant) cells were received from Dr. Arun Rishi (Wayne State University, Detroit, MI, USA). Rociletinib resistant H1975 (ROC-Res-H1975) cells were developed in our laboratory by continuous exposure of H1975 cells to rociletinib, starting from a dose of 150 nM and further escalating to 4 μM over a period of 8-10 months. All cells were grown in RPMI media supplemented with 10% FBS, 100 units per ml penicillin, and 100 mg/ml streptomycin under standard conditions of 5% CO 2 and 37˚C in a controlled humidified (95% relative humidity) incubator.
3D spheroid cell culture. Cells were cultured in RPMI media supplemented with heat inactivated FBS. Upon reaching 70% confluency, cells were routinely passaged using 0.05% trypsin-EDTA and collected according to standard cell culture procedures. Cells were then counted and reseeded on low attachment plates in heat inactivated media supplemented with 2% cultrex, under standard conditions of 5% CO 2 and 37˚C in a controlled humidified (95% relative humidity) incubator. After the formation of spheroids within 3 days, cells were subjected to different treatments. Media were changed every two days. h. Cell viability was assessed by using MTT assay as previously described (35).
Cell uptake studies. HCC827, HCC827-ERL-R, H1975, and ROC-Res-H1975 cells suspended in RPMI medium containing 2% cultrex reagent were seeded in Scivax low attachment 96-well plates and incubated at 37˚C and 5% CO 2 for 3 days before treatment. After the formation of spheroids, cells were pre-treated with Tel followed by incubation with FITC solution for 4 h. Cells were washed with PBS and then subjected to trypsinization using 0.25% trypsin EDTA solution for 5-10 min. Centrifugation was performed and the cell pellet was washed twice with PBS before resuspending at a density of 2×10 5 cells/ 500 μl in PBS for analysis using the BD FACS-Calibur flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA).
Western blot analysis. Whole cell protein lysates of HCC827, HCC827-ERL-R, H1975 and ROC-Res-H1975 cells treated with CFM 4.16 (10 μM), Tel (25 μM) and sorafenib (5 μM) either alone or in combinations were prepared by using RIPA buffer (Cell Signaling, Danvers, MA, USA) according to previously described methods (36). Protein estimation was carried out by using the bicinchoninic acid (BCA) assay. Briefly, 40 μg protein was loaded ANTICANCER RESEARCH 41: 4215-4228 (2021) into each well of a 10% SDS-PAGE gel (Mini-PROTEAN ® TGX™ Precast Gels). Protein was transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA) after electrophoresis. After transfer, membranes were blocked for 4-6 h using 3% bovine serum albumin in PBS containing 0.1% Tween-20 (PBST). Blots were then incubated with primary antibodies (1:1,000) overnight followed by washing thrice with PBST for 5 min each, and finally incubated with appropriate HRP-conjugated secondary antibodies [rabbit antimouse IgG (catalog number: 7076S; Cell Signaling Technology)]; goat anti-rabbit IgG (catalog number: 7074S; Cell Signaling Technology) for 1 h at room temperature. After washing with PBST again thrice for 5 min each, the blots were incubated with SuperSignal West Pico Chemiluminescent substrate and their images were captured using Chemidoc Instrument (Bio-Rad). The immunoblots were quantified by densitometry scanning using NIH ImageJ software. CFM 4.16 SNEDD formulation. CFM 4.16 NLF was prepared and characterized according to the methods previously described (37,38). Briefly, suitable quantity of CFM-4.16 was initially mixed with Compritol 888ATO, Miglyol 812N, and Geleol, followed by melting of this mixture at 70˚C to form a uniform and clear oil phase. The aqueous phase containing surfactant Tween 80 and D-α-tocopheryl polyethylene glycol succinate (Vitamin E TPGS) dispersed in double distilled water was added drop wise to the oil phase at 70˚C. Afterwards, NanoDebee was used for homogenising the coarse emulsion for 15 min under high pressure for about 5 cycles.
Rociletinib resistant H1975 xenograft model of non-small cell lung cancer. Female athymic nude mice (Foxn1 nu ; 3-5 weeks old, 20-25 grams' body weight, n=4) were procured from Envigo, housed in controlled climate conditions with a 12 h light/12 h dark cycle with free access to water and food intake, and acclimatized for 7-10 days. All the animal experiments carried out were reviewed and approved by the Institutional Animal Use and Care Committee of Florida Agricultural and Mechanical University and carried out according to the NIH guidelines (Guide for the care and use of laboratory animals) and applicable national laws. All animal experiments complied with the ARRIVE guidelines (39). Rociletinib resistant H1975 xenograft model of NSCLC was developed by subcutaneous injection of 3 million ROC-Res-H1975 cells, suspended in matrigel in 1:1 ratio into the right flank of nude mice. The digital Vernier caliper instrument was used for measuring the length and width of the tumors. Tumor volume (TV) was then calculated by using the formula TV=½ ab 2 , where 'a' and 'b' represent the length and width of the tumors, respectively. After the animals showed a tumor volume of around 400 mm 3 , they were randomly divided into six groups. Animals were treated with Tel (10 mg/kg body weight), CFM4.16 NLF (40 mg/kg body weight) and sorafenib (30 mg/kg body weight) alone for 2 weeks. For CFM 4.16 and sorafenib combination, animals were pre-treated with CFM 4.16 followed by treatment with sorafenib for 2 weeks. For Tel, CFM 4.16 and sorafenib combination, animals were pre-treated with Tel (10 mg/kg body weight) thrice a week followed by treatment with CFM4.16 NLF (40 mg/kg body weight) and sorafenib (30 mg/kg body weight) for 2 weeks. Tumor volume was measured twice a week during the duration of drug treatments.
Statistical analysis. All the data are presented as the mean±standard error of mean (SEM). Significant difference among the treatment groups was determined by using either Student's t-test or one-way ANOVA through using GraphPad prism version 5.0 (San Diego, CA, USA). A p-Value less than 0.05 was considered to be statistically significant.

Results
Combinatorial anticancer effects of sorafenib, CFM 4. 16 and telmisartan in NSCLC and drug resistant NSCLC cells Cytotoxicity assays were performed to determine initially the individual anti-cancer potential of sorafenib, CFM 4.16 and Tel in 2D and 3D cultures of HCC827, erlotinib resistant HCC827 (HCC827-ERL-R), H1975 and rociletinib resistant H1975 (ROC-Res-H1975) cells. The same number of cells (8×10 3 cells/well) was used for comparing cytotoxic data in 2D and 3D cultures. CFM 4.16 and Tel were treated with concentrations ranging from 6.25 to 100 μM. Sorafenib concentrations ranged from 0.78 to 100 μM. We observed a dose-dependent decrease in the viability of all tested cell lines with the treatments after 48 h. IC 50 values of sorafenib in 3D cultures of HCC827, HCC827-ERL-R, H1975 and ROC-Res-H1975 cells were found to be 5.77-fold, 3.73-fold, 3.30-fold, and 3.18-fold higher when compared to 2D cultures of these cells, respectively (Table I). IC 50 values of CFM 4.16 in 3D cultures of HCC827, HCC827-ERL-R, H1975 and ROC-Res-H1975 cells were found to be 2.27-fold, 2.49-fold, 1.98-fold, and 2.05-fold higher when compared to 2D cultures of these cells, respectively (Table I). IC 50 values of Tel in 3D cultures of HCC827, HCC827-ERL-R, H1975 and ROC-Res-H1975 cells were found to be 1.67-fold, 1.67-fold, 1.34-fold, and 1.28-fold higher when compared to 2D cultures of these cells, respectively (Table I). IC 50 values in 3D cultures with all the treatments were higher in comparison to 2D monolayers, suggesting the influence of the 3D tumor microenvironment in determining anti-cancer efficacy. Based on our cytotoxicity assay observations in 2D and 3D cultures, we selected 10 μM of CFM 4.16 and 25 μM of Tel for combination studies with sorafenib. CFM 4.16 pre-treatment for 12 h increased the sensitivity of sorafenib in both 2D and 3D cultures of HCC827, HCC827-ERL-R, H1975 and ROC-Res-H1975 cells (Table I). Tel and CFM 4.16 combination pre-treatment for 12 h did not lead to an improved sensitivity to sorafenib in 2D cultures of HCC827, HCC827-ERL-R, H1975 and ROC-Res-H1975 cells when compared to the IC 50 values of CFM 4.16 and sorafenib combination (Table I). However, Tel and CFM 4.16 pre-treatment significantly improved the sensitivity of sorafenib in 3D cultures of all the tested cell lines ( Table I), suggesting that tumor stromal disruption by Tel was responsible for the higher anti-cancer effects of the combination treatment in 3D cultures.  Tel, CFM 4.16 NLF and sorafenib combination displays superior anti-cancer effect in rociletinib resistant H1975 NSCLC xenograft model. As CFM 4.16, sorafenib and Tel combination showed good anti-cancer effects in H1975 and drug resistant H1975 cells in vitro, we further investigated their combinatorial effects in rociletinib resistant H1975 NSCLC xenograft model to ensure efficacy. We developed rociletinib resistant H1975 cells in our laboratory by continuous exposure of H1975 cells to rociletinib, starting from a dose of 150 nM, and further escalating to 4 μM over a period of 8-10 months. H1975-rociletinib resistant NSCLC xenograft model was developed by subcutaneous injection of rociletinib resistant H1975 cells on to the right side of nude mice. Tumor volume was checked twice weekly, once the palpable tumor was observed. The tumours were allowed to grow to the desired tumor volume (i.e., approximately around 400 mm 3 ), and thereafter we divided the animals into five groups (n=6), so as to minimize the weight and tumour size difference among the groups. Control mice received 0.9% ANTICANCER RESEARCH 41: 4215-4228 (2021) 4218 Table I. Cytotoxicity of sorafenib, CFM 4. 16 Figure 6). We also observed that CFM 4.16, sorafenib and Tel combination significantly decreased the expression of TGF-β (p<0.001), MMP-9 (p<0.001), and lung cancer stem cell markers (p<0.001), which contribute to the sorafenib poor uptake and resistance in lung cancer cells ( Figure 6). The combination therapy also significantly increased the expression of E-cadherin (p<0.001) in the ROC-Res-H1975 xenograft model of NSCLC (Figure 6).

Discussion
Lung cancers with epidermal growth factor receptor (EGFR) gene mutations account for approximately 40% of lung adenocarcinomas in East Asians and around 20% in Caucasians and African Americans (41). TKIs are considered as the first line chemotherapy for NSCLC. Resistance to TKIs such as gefitinib, erlotinib or sorafenib involves hyperactivation/mutations in oncogenic proteins such as EGFR, MET, vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), BRAF, Alk, and Src in various cancers (42)(43)(44). Different EGFR-dependent and EGFR-independent mechanisms have been demonstrated to be responsible for the development of third generation TKI (i.e., osimertinib and rociletinib) resistance in EGFR mutant NSCLC (45)(46)(47). Inhibition of AKT (i.e., by using MK-2206 and GDC-0068) and VEGFR/MET/AXL axis (i.e., by XL-880) have been reported to improve the sensitivity of rociletinib in rociletinib resistant models of NSCLC (14). Due to the paucity of therapeutic options readily available for secondary resistance after rociletinib treatment, there is an urgent unmet clinical challenge for the identification of therapeutics to combat rociletinib resistance in NSCLC. To the best of our knowledge, this study investigated for the first time the anticancer potential of telmisartan (Tel), CFM 4.16 and sorafenib combination in both in vivo and in vitro models of rociletinib resistant H1975 cells.
Herein, we investigated the anti-cancer efficacy of sorafenib, Tel, CFM 4.16, CFM 4.16 + sorafenib combination, and Tel + CFM 4.16 + sorafenib combination in 2D and 3D spheroid cultures of HCC827, erlotinib resistant HCC827, H1975 and rociletinib resistant H1975 cells. We observed higher IC 50 values in 3D spheroid cultures when compared to 2D cultures of these cells, indicating the influence of the tumor microenvironment in determining the anti-cancer effects of chemotherapeutics. 3D spheroids serve as excellent physiologic tumor models in mimicking in vivo the tumour microenvironment for evaluating the anticancer effects of various chemotherapeutics (48,49).
Multiple evidence has demonstrated the low permeability of free FITC across the cell membranes (50,51). Our cell viability data has shown that TEL + CFM 4.16+ sorafenib combination decreased the proliferation of 3D spheroids. This is further supported by a cell uptake study, which showed that Tel pre-treatment significantly increased the uptake of FITC in 3D spheroids when compared to free FITC stained 3D spheroids as analyzed by flow cytometry. Decreased diffusion of chemotherapeutics through the spheroids contribute to resistance in 3D spheroid cultures, and tumor penetration remains a vital barrier inhibiting treatment response (52).
Rociletinib resistant H1975 xenograft model was developed by subcutaneous injection of rociletinib resistant H1975 cells into the right flank of nude mice. It was observed that Tel + CFM 4.16 NLF + sorafenib combination significantly decreased tumor volume when compared to control.
To elucidate the molecular changes involved behind the enhanced anticancer effects of Tel + CFM 4.16 + sorafenib combination in our study, we investigated the protein expression of p-EGFR/EGFR and p-MET/MET in 2D cultures of wild type and rociletinib resistant H1975 cells. It was observed that telmisartan (Tel), CFM 4.16 and sorafenib combination decreased p-MET/MET expression in both wild type and rociletinib resistant H1975 cells. However, this combination significantly decreased p-EGFR/EGFR in rociletinib resistant H1975 cells when compared to wild type H1975 cells. This demonstrates the efficacy of combination therapy in rociletinib resistant H1975 cells. Moreover, this combination therapy significantly decreased the protein expression of p-EGFR/EGFR and p-MET/MET in rociletinib resistant H1975 xenograft model, indicating its therapeutic efficacy. The superior effects of this combination in rociletinib resistant H1975 xenograft model may be due to the disruption of tumor stromal barriers by telmisartan (i.e., an anti-fibrotic agent), which facilitates CFM 4.16 and sorafenib to penetrate more into the deeper layers of tumors, to produce more anticancer effects when administered in vivo.
Cancer stem cells (CSCs) are involved in the initiation, maintenance, progression, metastasis and drug resistance of cancer cells (53). Various factors such as EMT, alterations in the metabolism of tumor cells, epigenetic modifications, and the tumor microenvironment are involved in the resistance of cancer cells through activation of various signaling pathways associated with cancer stemness (54). TGF-β is involved in the transformation of cells from a non-CSC to an CSC phenotype through activation of Zinc finger E-boxbinding homeobox 1 (ZEB1), a key mediator of EMT (55). EMT is well demonstrated to be related with the stemness and maintenance of CSCs, which display stem cell-like properties by expressing Sox2, Oct4 and Nanog genes (56). Deregulated EGFR is involved in CSCs stemness and drug resistance of cancer cells (57). RAF/MEK/ERK/STAT3/AKT signaling is also associated with stemness of CSCs, drug resistance and recurrence of cancers (58). CSCs stemness (i.e., increased expression of Sox2 and CD24) is one of the factors responsible for resistance of cancer cells to BRAF inhibitors (59). A study by Cheriyan et al., has demonstrated that CARP-1 functional mimetic (CFM 4.16) in combination with sorafenib significantly decreased tumor growth of rociletinib-resistant H1975 NSCLC cells by inducing CARP-1 expression and facilitating apoptosis (22). Herein, we investigated the expression of CSC markers such as Nanog, Oct4 and Sox2 and observed that Tel, CFM 4.16 and sorafenib combination significantly decreased the levels of these proteins in 2D cultures of wild type and rociletinib resistant H1975 cells as well as in rociletinib resistant H1975 tumor cell lysates. This suggests the efficacy of this combination in decreasing drug resistance and improving anti-cancer effects.  Matrix metalloproteinases (MMPs) are involved in the pathophysiology of several cancers through zinc-dependent degradation and remodeling of extracellular matrix (ECM) proteins (60,61). MMP-9 plays a crucial role in the invasion, metastasis, angiogenesis and progression of various cancers (62). It is negatively correlated with immune regulation of cancers through transforming growth factor-β (TGF-β) activation and shedding of intercellular adhesion molecule-1 (ICAM-1). MMP-9 is over-expressed and considered as a biomarker for the prognosis of breast cancer (63). TGF-β mediates not only the proliferation, differentiation, apoptosis, migration, adhesion, immune surveillance, and survival of cancer cells, but also favors the production of extracellular matrix proteins (64,65). Tel, CFM 4.16 NLF and sorafenib combination significantly decreased the protein expression of MMP-9 in rociletinib resistant H1975 tumor model.
Decreased expression of E-cadherin, a well-known tumor suppressor and epithelial cell-cell adhesion molecule is associated with loss of differentiation and increased EMT in various cancers (66,67). E-cadherin serves as a prognostic marker in breast, endometrial, prostate, gastric and lung cancers (68)(69)(70)(71)(72). A study by Wang et al., has demonstrated that TGF-β induces morphological and phenotypic changes associated with increased EMT and decreased E-cadherin levels (73). However, it was recently reported that loss of E-cadherin causes up-regulation of TGF-β signaling related genes in breast cancer cells (74). E-cadherin suppresses tumorigenicity and metastasis through inhibition of β-catenin and EGFR proteins, which are oncogenic in nature (75,76). Tel, CFM 4.16 NLF and sorafenib combination significantly increased the protein expression of E-cadherin in rociletinib resistant H1975 tumor model. This indicates that the combination therapy not only decreases migration and invasion, but also increases the tumor-suppressive effects in the rociletinib resistant H1975 xenograft model.

Conclusion
In summary, the combination of Tel, CFM 4.16 and sorafenib displays superior anti-cancer effects in 2D and 3D spheroid cultures of rociletinib resistant H1975 cells, along with an excellent therapeutic potential in decreasing tumor growth in a rociletinib resistant H1975 xenograft model of NSCLC by decreasing the protein expression of p-EGFR/EGFR, p-MET/MET, CSC markers (Nanog, Sox2 and Oct4), TGF-β (i.e., involved in fibrosis) and MMP9 (involved in migration) and increasing the expression of E-cadherin (i.e., a tumor suppressor and involved in EMT). This study provides preliminary insights of the therapeutic efficacy of Tel, CFM 4.16 and sorafenib combination in decreasing rociletinib resistance of NSCLC.

Conflicts of Interest
The Authors declare that they have no conflicts of interest associated with this study.