Co-targeting of Cyclooxygenase-2 and FoxM1 is a viable strategy in inducing anticancer effects in colorectal cancer cells
- Maqbool Ahmed†1,
- Azhar R Hussain†1,
- Abdul K. Siraj1,
- Shahab Uddin1,
- Nasser Al-Sanea2,
- Fouad Al-Dayel3,
- Mohammed Al-Assiri4,
- Shaham Beg1 and
- Khawla S. Al-Kuraya1, 5Email author
© Ahmed et al. 2015
Received: 18 March 2015
Accepted: 1 July 2015
Published: 10 July 2015
Cross-talk between deregulated signaling pathways in cancer cells causes uncontrolled growth and proliferation. These cancers cells become more aggressive and quickly develop resistance to therapy. Therefore targeting of these deregulated pathways simultaneously can result in efficient cell death of cancer cells. In this study we investigated co-expression of Cox-2 and FoxM1 in a cohort of colorectal carcinoma (CRC) samples and also examined whether inhibition of Cox-2 and FoxM1 simultaneously can lead to inhibition of cell viability and induction of apoptosis in colorectal cancer cell lines and in vivo xenografts.
Protein expression of Cox-2 and FoxM1 was determined in a large cohort of 770 clinical CRC samples in a tissue micro-array format by immunohistochemistry. Cell death was measured using live dead assay. Apoptosis was measured by annexin V/PI dual staining. Immunoblotting was performed to examine the expression of proteins. Calcusyn software was utilized to estimate the synergistic doses using chou and Talalay method.
Co-expression of Cox-2 and FoxM1 was detected in 33.3 % (232/697) of CRC’s and associated with an aggressive phenotype characterized by younger age (p = 0.0191), high proliferative index marker; Ki-67 (p = 0.004) and MMP-9 (p = 0.0116) as well as activation of AKT (p = 0.0214). In vitro, inhibition of FoxM1 and Cox-2 with pharmacological inhibitors; Thiostrepton and NS398 resulted in efficient down-regulation of FoxM1 and Cox-2 expression along with in-activation of AKT and inhibition of colony formation, invasion and migratory capability of CRC cells. In addition, there was also inhibition of cell viability and induction of apoptosis via the mitochondrial apoptotic pathway in CRC cell lines. Finally, treatment of CRC xenograft tumors in nude mice with combination of Cox-2 and FoxM1 inhibitors inhibited tumor growth significantly via down-regulation of Cox-2 and FoxM1 expression.
These findings demonstrate that co-expression of Cox-2 and FoxM1 might play a critical role in the pathogenesis of CRC. Therefore, targeting of these pathways simultaneously with sub toxic doses of pharmacological inhibitors can be a potential therapeutic approach for the treatment of this subset of CRC.
KeywordsColorectal cancer Cox-2 FoxM1 Cell viability Colony formation Invasion
Despite increased awareness in the general population regarding colorectal cancer (CRC), it still remains a major cause of mortality and morbidity worldwide . This increase has been attributed to a combination of environmental and genetic factors in the general population [2, 3]. Even though CRC is very well studied along with established diagnostic markers, most of CRC cancers present at late stages of disease and therefore have a poor prognosis . In addition, recurrence and metastasis of CRC also carry a very high mortality rate . Therefore, there is a need for improvement in the diagnosis of CRC as well as identification of newer therapeutic targets that can be specifically drugged to improve the management of these cancers.
An important key survival molecule that is currently being investigated as a molecular marker and a potential therapeutic target is cyclooxygenase-2 (Cox-2) in various cancers. The main function of Cox is to synthesize prostaglandins from arachidonic acid . There are two isoforms of Cox; Cox-1 that is found to be expressed in normal cells  and Cox-2 that is preferentially expressed in cancer cells  and its expression is enhanced by pro-inflammatory cytokines and carcinogens [8, 9]. Cox-2 has been found to be over-expressed by us and others in a variety of cancer including breast, ovary, colorectal, thyroid and lung [10–14]. Prophylactic use of Cox-2 inhibitors such as aspirin has been shown to decrease the incidence of certain cancers [15–18].
Forkhead box protein M1 (FoxM1) is a member of FoxM family that consists of more than 50 proteins that are characterized by a conserved 100 amino acid DNA binding domain [19, 20]. FoxM1 has also been known to regulate the transcriptional activity of number of genes including cyclin B, cyclin A and Aurora B kinase, which are very important for cell cycle progression and mitotic entry [21–23]. Loss of FoxM1 expression has also been reported to generate mitotic spindle defects leading to mitotic catastrophe [21, 24, 25]. FoxM1 signaling has been implicated to be associated with carcinogenesis of tumor development in CRC as well as other solid tumors [22, 26–33].
A number of dysregulated survival pathways have the ability to cross-talk with each other to increase aggressiveness of various cancers [34, 35]. These cross-talks allow the cancer cell to avoid different in vivo and in vitro threats thereby allowing un-supervised growth and proliferation and the cancers cells become more aggressive and quickly develop resistance to therapy . Inhibiting one pathway may not be enough to elicit a complete response because of the cross-talk with other pathways thereby eliciting a feedback response to reactivate the targeted pathway . Targeting multiple pathways also helps in decreasing drug-induced toxicity by using sub-toxic doses in combination.
There have been many studies performed to investigate the role of Cox-2 and FoxM1 in tumorigenesis independently however there are only few studies where these molecules are studied together . Therefore, in this study, we first investigated co-expression of Cox-2 and FoxM1 in CRC clinical samples followed by determining whether targeting of co-expression of FoM1 and Cox-2 can generate efficient anticancer effects in CRC cells both in vitro as well as in vivo models.
Evaluation of molecular expression of Cox-2 and FoxM1 in CRC tissues
Correlation of Cox-2 & Fox-M1 co-expression with clinico-pathological parameters in colorectal carcinomaa
Total number of cases
OS 5 Years
Inhibition of Cox-2 and FoxM1 causes inhibition of cell viability in CRC cell lines
Synergistic activity of thiostrepton and NS398 in CRC cell lines
As our data showed FoxM1 and Cox-2 co-expression was present in CRC, we hypothesized that targeting of FoxM1 and Cox-2 expression together can lead to efficient cytotoxic effects in CRC cells. Therefore we sought to determine whether co-treatment of CRC cell lines with Thiostrepton, and NS398 at sub-toxic doses, can potentiate anticancer effects in CRC cells.
Co-treatment of CRC cells with thiostrepton and NS398 induces apoptosis via mitochondrial apoptotic pathway
Inhibition HT29 xenografts by combinational treatment of thiostrepton and NS398 in nude mice
In this study, we have investigated the role of Cox-2 and FoxM1 co-expression in a large cohort of 770 Middle Eastern CRC in a tissue microarray format to determine the protein expression by immunohistochemistry. It has been previously shown that Cox-2 and FoxM1 expression are associated with a poor prognosis in CRC [45, 46]. In this study, we found a significant association between FoxM1 and Cox-2 over-expression in Middle Eastern CRC samples (p = 0.0115). Co-expression of FoxM1 and Cox-2 was also found to be associated with an aggressive phenotype that was characterized by younger age (p = 0.0191), proliferative marker Ki-67 (0.0004), MMP-9 (p = 0.0116) and activation of AKT (p = 0.0214). These data reiterates the point that targeting these two molecules simultaneously using small molecular inhibitors may be more beneficial for the management of this aggressive phenotype of CRC when compared to treatment with single agent.
Cross talk between survival pathways is slowly emerging as one of the leading causes of drug resistance to small molecular inhibitors for the treatment of cancer. After an initial response to the treatment, resistance to therapy quickly develops due to re-activation of the target molecule either by up-stream pro-survival pathway molecules or negative feed-back mechanism by down-stream molecules. To counteract this resistance, recent studies have shown targeting multiple pro-survival molecules of different survival pathways that are associated with each other with combination of specific inhibitors simultaneously is more beneficial than treatment with single agent alone [47, 48]. Combination treatment is also beneficial because the dose of each drug is considerably decreased when used in combination thereby reducing the chances of toxicity to normal cells. Therefore, the role of single agent treatment with molecular inhibitors is diminishing and targeting various cancers with multiple inhibitors is on the rise.
A strong association between expression of FoxM1 and Cox-2 with MMP-9 expression has been reported in a number of cancers [31, 49, 50]. Previously, it has also been reported in lung cancer that transcriptional depletion of FoxM1 expression can cause reduced Cox-2 expression and on the other hand, induced over-expression of FoxM1 protein can increase Cox-2 promoter activity . These findings are in conco rdance with our data where pharmacological and transcriptional inhibition of FoxM1 expression down-regulates the expression of Cox-2. Furthermore, our data also demonstrates that depletion of Cox-2 expression does not affect FoxM1 expression suggesting that FoxM1 is functional upstream of Cox-2. Combined targeting of Cox-2 and FoxM1 with pharmacological inhibitors also depletes colony formation as well as invasive and migratory capabilities of CRC cells in vitro via down-regulation of MMP-9 thereby indicating the utility of combined targeting of these molecules for inhibition of metastasis in CRC cells.
Combined targeting of Cox-2 and FoxM1 not only inhibits the invasive and migratory capability of CRC cells, they also cause inhibition of cell viability and induction of apoptosis. This effect occurs via inactivation of an important survival molecule; p-AKT, that plays an important role in the survival of cancer cells and is found to be constitutively activated in various cancers . Our data showed that dephosphorylation of AKT led to activation of the mitochondrial apoptotic pathway initiated by Bax conformational changes and translocation to the mitochondrial membrane, thereby leading to changes in the mitochondrial membrane potential and finally activation and cleavage of caspases. Once caspases are activated, there is cleavage of PARP; an essential enzyme that is required for repairing single stranded breaks in DNA  and is a hallmark of cells undergoing apoptosis. Our in vivo studies further validate our hypothesis that co-treatment of mice bearing palpable CRC xenograft with Thiostrepton and NS398 leads to regression of tumor growth via down-regulation of FoxM1, Cox-2, MMP-9, inactivation of AKT and cleavage of caspase-3 which is consistent with our in vitro findings.
Altogether, we found that 33.3 % of CRC clinical samples co-express Cox-2 and FoxM1 and this sub-group is associated with an aggressive phenotype. Therefore, our data highlights the importance of co-targeting of deregulated survival pathways (Cox-2 and FoxM1) in CRC cells can lead to anticancer effects. Our data showed that combination treatment of CRC cells with sub-optimal doses of Thiostrepton and NS398 caused functional inhibition of Cox-2 and FoxM1 simultaneously. Even though, Thiostrepton and NS398 have been previously shown to be effective in suppressing growth and inducing apoptosis in CRC cells at higher concentrations [31, 52], this study emphasizes the importance of targeting multiple survival molecules with sub-optimal doses of Thiostrepton and NS398 to successfully inhibit cell growth, invasion, migration and induce apoptosis in CRC. Further studies are warranted to further investigate the utility of combination treatment with Thiostrepton and NS398 for the treatment of CRC in clinical settings.
Material and methods
Patient selection and tissue microarray construction
Seven hundred and seventy patients with CRC diagnosed between 1990 and 2011 were selected from King Faisal Specialist Hospital and Research Centre. Clinical and histopathological data were available for all these patients. Colorectal Unit, Department of Surgery, provided long-term follow-up data. Patients with colon cancer underwent surgical colonic resection and rectal cancer underwent anterior resection or abdominoperineal resection. Majority of node positive colon cancers received 5-fluorouracil based adjuvant chemotherapy. A vast majority of the rectal cancers received radiotherapy alone or chemo-radiotherapy prior to surgery followed by adjuvant chemotherapy after surgery. Tissue microarrays were constructed from formalin-fixed, paraffin-embedded colorectal carcinoma specimens as described previously . Institutional Review Board (IRB) of the King Faisal Specialist Hospital & Research Centre approved the study (NSTIP 10-BIO-959-20 and RAC 2140 005).
TMA slides were processed and stained manually as described previously . Sections were deparaffinized in xylene and rehydrated through graded alcohol to water. Antigen retrieval was done in a Pascal Pressure cooker at 120 °C for 8 min using the Dako Retrieval solution, pH 6 (S2369; Dako Cytomation, Copenhagen, Denmark). Endogenous peroxidase activity was blocked by incubating the slides in 3 % H2O2 in water for 30 min at room temperature. Sections were incubated in 1 % BSA for 30 min then wiped off and dilution of Cox-2 and FoxM1 was applied on the slides and incubated overnight at room temperature. Subsequently sections were incubated with Envision + secondary antibody for 1 h at room temperature and visualization was done using the liquid DAB + substrate chromogen system. Only fresh cut slides were stained simultaneously to minimize the influence of slide ageing and maximize repeatability and reproducibility of the experiment. Details of primary antibodies used, dilutions, cut-off and incidences of positive cases are listed in Additional file 9: Table S5. H-score was used for categorizing the expression of Cox-2 and FoxM1. Each TMA spot was assigned an intensity score from 0 to 3 (I0, I1–I3) and proportion of the tumor staining for that intensity was recorded in 5 % increments from 0 to 100 (P0, P1–P3). A final H score (range, 0–300) was obtained by adding the sum of scores obtained for each intensity I and proportion of area stained. X-tile plots were constructed for assessment of biomarker and optimization of cutoff points based on outcome, as described previously . The CRCs were stratified into two groups based on X-tile plots: one with complete absence or reduced staining and the other with overexpression. For Ki-67 cut-off of ≥50 % nuclear staining was used and for p-AKT intensity score 2+/3+ was considered as positive.
Contingency table analysis and χ 2 tests were used to study relationship between clinicopathological variables and gene amplification. The limit of significance for all analyses was defined as a P value of 0.05; two-sided tests were used in all calculations. The JMP 10.0 software package (SAS Institute, Cary, NC) was used for data analyses.
Reagents and antibodies
Thiostrepton (FoxM1 selective inhibitor)  was purchased from Tocris Cookson Inc (Ellisville, MO). NS398 (COX-2 inhibitor) was purchased from Caymen chemical company, (Ann Arbor, MI). Antibodies against cleaved caspase-3, Cox-2, AKT and p-AKT antibodies were purchased from Cell Signaling Technologies (Beverly, MA). FoxM1, Bax, Beta-actin, caspase-3 and poly (ADP) ribose polymerase (PARP) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). MMP-9 antibodies were purchased from Anespec, (San Jose, CA). Annexin V/PI kit was purchased from Molecular Probes (Eugene, OR, USA).
HT29, DLD1, LOVO, HCT-15 and Caco-2 were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. All cell lines were tested for immunological markers and cytogenetics. The cell lines were also fingerprinted and species was confirmed by IEF of AST, MDH and NP. Cells were cultured in RPMI 1640 medium supplemented with 10 % (vol/vol) fetal bovine serum, 100 U/ml Penicillin and 100 U/ml Streptomycin at 37 °C in humidified atmosphere containing 5 % CO2. All the experiments were performed in RPMI-1640 containing 5 % fetal bovine serum.
Cell growth studies by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays
104 cells were incubated in triplicate in a 96-well plate in a final volume of 0.2 ml for 48 h at 37 °C. Cell viability assay using MTT was performed as described previously .
Live dead assay
To determine cell death, Live-Dead assay (Invitrogen, Eugene, OR) was used as described by the manufacturer. HT29 and Caco-2 cells were treated either alone with NS398, and Thiostrepton or in combination as described in the legends. Following incubation for 48 h, cells were suspended in 1 ml PBS containing 50 mM calcein AM and 8 mM ethidium homodimer and cells were incubated in the dark for 20 min. 50 μl of suspension was transferred on slides and visualized under an Olympus fluorescent microscope using a longpass filter.
Soft agar colony assay
Soft agar colony experiments were performed according to the manufacturer’s protocol (Cheminon International, Temecula, CA, USA). Briefly, after treatment of cells with NS398, Thiostrepton or a combination of the two inhibitors for 48 h, 2500 cells were plated in 0.5 ml culture medium containing 0.4 % (v/v) top agar and 20 % fetal bovine serum (FBS) layered over a basal layer of 0.8 % (v/v) agar and 20 % FBS with culture medium and allowed to grow for 4 weeks as described previously (our REF). Following 4 weeks incubation, cells were stained at a final concentration of 1 mg/ml cell stain solution that was supplied with the kit.
Cell invasion and migration assay
Cell invasion and migration assay were performed using 24-well Transwell Permeable Supports with 8-lM pores (Corning, Lowell, MA). Briefly, after treatment of cells with NS398, Thiostrepton or a combination of the two inhibitors for 48 h, cells were harvested, counted again and 1.25 x 105 cells were suspended in serum-free medium and seeded into Transwell inserts either uncoated (for migration assay) or coated (for invasion assay) with growth factor-reduced Matrigel (BD Biosciences, Bedford, MA). Bottom wells were filled with complete media for 24 h. After incubation of 24 h, filters containing the cells were stained with Diff-Quick stain set (Fisher Scientific, Pittsburg, PA), photographed under a fluorescent microscope and manual cell counts were obtained .
Gene Silencing using siRNA
FoxM1 siRNA, Cox-2 siRNA and Scrambled control siRNA were purchased from Qiagen (Valencia, CA, USA). Cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for 6 h following which the lipid and siRNA complex was removed and fresh growth medium was added. Cells were lysed 48 h after transfection and specific protein levels were determined by Western Blot analysis with specific antibodies.
Annexin V/PI dual staining
HT29 and Caco-2 cells were treated either with NS398, Thiostrepton or in combination as described in the legends. For detection of apoptosis, cells were harvested and percentage apoptosis was measured by flow cytometry after staining with flourescein-conjugated annexin-V and propidium iodide (PI) (Molecular probes, Eugene, OR) .
Measurement of mitochondrial membrane potential
Cells were treated with NS398 and Thiostrepton as described in the legends for 48 h, washed twice with PBS, and re-suspended in mitochondrial incubation buffer. JC1 staining and flow cytometry were done as described previously.
Cell lysis and immunoblotting
Cells were lysed as previously described . Proteins were immunoblotted with different antibodies and visualized by the enhanced chemiluminescence (Amersham, Piscataway, NJ) method.
Detection of Bax conformational changes
Detection of Bax conformation was performed as previously described . In brief, HT29 cells were treated with combination of 10 μM NS398 and 5 μM Thiostrepton for indicated time periods after which proteins were lysed and immunoblotted using N20 Bax polyclonal antibody.
Animals and xenograft study
Six weeks old nude mice were obtained from Jackson Laboratories (Maine, USA) and maintained in a pathogen free animal facility at least 1 week before use. All animal studies were done in accordance with institutional guidelines. For Xenograft study, mice were inoculated sub-cutaneously into the right abdominal quadrant with 10x106 cells of HT29 in 200 μL PBS. After 1 week, mice were randomly assigned into four groups: The first group received DMSO. The three groups received N398 (15 mg/kg), Thiostrepton (150 mg/kg) and combination of 15 mg/kg NS398 and 150 mg/kg Thiostrepton, intra-peritoneally respectively. Mice were given these drugs twice weekly. The body weight and tumor volume of each mouse was monitored weekly. The tumor volume was measured as described previously . After 5 weeks treatment, mice were sacrificed and individual tumors were weighed, then snap-frozen in liquid nitrogen for storage.
We would like to acknowledge the efforts of Dr. Sally Al Abdulmohsen, Saravanan Thangavel and Saeeda Ahmed for their technical assistance.
This study was supported by King AbdulAziz Centre for Science and Technology with grant number NSTIP10-BIO959-20.
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