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.
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.
- Ross JS, Torres-Mora J, Wagle N, Jennings TA, Jones DM. Biomarker-based prediction of response to therapy for colorectal cancer: current perspective. Am J Clin Pathol. 2010;134:478–90.PubMedView ArticleGoogle Scholar
- Chan AT, Giovannucci EL. Primary prevention of colorectal cancer. Gastroenterology. 2010;138:2029–43.PubMed CentralPubMedView ArticleGoogle Scholar
- Imamura Y, Lochhead P, Yamauchi M, Kuchiba A, Qian ZR, Liao X, et al. Analyses of clinicopathological, molecular, and prognostic associations of KRAS codon 61 and codon 146 mutations in colorectal cancer: cohort study and literature review. Mol Cancer. 2014;13:135.PubMed CentralPubMedView ArticleGoogle Scholar
- Worthley DL, Leggett BA. Colorectal cancer: molecular features and clinical opportunities. Clin Biochem Rev. 2010;31:31–8.PubMed CentralPubMedGoogle Scholar
- Segal NH, Saltz LB. Evolving treatment of advanced colon cancer. Annu Rev Med. 2009;60:207–19.PubMedView ArticleGoogle Scholar
- Meyer-Kirchrath J, Schror K. Cyclooxygenase-2 inhibition and side-effects of non-steroidal anti-inflammatory drugs in the gastrointestinal tract. Curr Med Chem. 2000;7:1121–9.PubMedView ArticleGoogle Scholar
- Zhu YM, Azahri NS, Yu DC, Woll PJ. Effects of COX-2 inhibition on expression of vascular endothelial growth factor and interleukin-8 in lung cancer cells. BMC Cancer. 2008;8:218.PubMed CentralPubMedView ArticleGoogle Scholar
- Zhu Z, Zhong S, Shen Z. Targeting the inflammatory pathways to enhance chemotherapy of cancer. Cancer Biol Ther. 2011;12:95–105.PubMedView ArticleGoogle Scholar
- Anto RJ, Mukhopadhyay A, Shishodia S, Gairola CG, Aggarwal BB. Cigarette smoke condensate activates nuclear transcription factor-kappaB through phosphorylation and degradation of IkappaB (alpha): correlation with induction of cyclooxygenase-2. Carcinogenesis. 2002;23:1511–8.PubMedView ArticleGoogle Scholar
- Howe LR. Inflammation and breast cancer. Cyclooxygenase/prostaglandin signaling and breast cancer. Breast Cancer Res. 2007;9.Google Scholar
- Uddin S, Ahmed M, Hussain A, Assad L, Al-Dayel F, Bavi P, et al. Cyclooxygenase-2 inhibition inhibits PI3K/AKT kinase activity in epithelial ovarian cancer. Int J Cancer. 2010;126:382–94.PubMedView ArticleGoogle Scholar
- Moreira L, Castells A. Cyclooxygenase as a target for colorectal cancer chemoprevention. Curr Drug Targets. 2011;12:1888–94.PubMedView ArticleGoogle Scholar
- Krawczyk-Rusiecka K, Wojciechowska-Durczynska K, Cyniak-Magierska A, Adamczewski Z, Galecka E, Lewinski A. COX-2 expression in papillary thyroid carcinoma (PTC) in cytological material obtained by fine needle aspiration biopsy (FNAB). Thyroid Res. 2011;4:3.PubMed CentralPubMedView ArticleGoogle Scholar
- Kim SJ, Rabbani ZN, Dong F, Vollmer RT, Schreiber EG, Dewhirst MW, et al. Phosphorylated epidermal growth factor receptor and cyclooxygenase-2 expression in localized non-small cell lung cancer. Med Oncol. 2010;27:91–7.PubMed CentralPubMedView ArticleGoogle Scholar
- Avivi D, Moshkowitz M, Detering E, Arber N. The role of low-dose aspirin in the prevention of colorectal cancer. Expert Opin Ther Targets. 2012;16 Suppl 1:S51–62.PubMedView ArticleGoogle Scholar
- Lo-Ciganic WH, Zgibor JC, Bunker CH, Moysich KB, Edwards RP, Ness RB. Aspirin, nonaspirin nonsteroidal anti-inflammatory drugs, or acetaminophen and risk of ovarian cancer. Epidemiology. 2012;23:311–9.PubMed CentralPubMedView ArticleGoogle Scholar
- McCarty MF. Minimizing the cancer-promotional activity of cox-2 as a central strategy in cancer prevention. Med Hypotheses. 2012;78:45–57.PubMedView ArticleGoogle Scholar
- Printz C. Celecoxib may prevent lung cancer. Cancer. 2012;118:3.Google Scholar
- Carlsson P, Mahlapuu M. Forkhead transcription factors: key players in development and metabolism. Dev Biol. 2002;250:1–23.PubMedView ArticleGoogle Scholar
- Kaestner KH, Knochel W, Martinez DE. Unified nomenclature for the winged helix/forkhead transcription factors. Genes Dev. 2000;14:142–6.PubMedGoogle Scholar
- Wang X, Kiyokawa H, Dennewitz MB, Costa RH. The Forkhead Box m1b transcription factor is essential for hepatocyte DNA replication and mitosis during mouse liver regeneration. Proc Natl Acad Sci U S A. 2002;99:16881–6.PubMed CentralPubMedView ArticleGoogle Scholar
- Kalin TV, Wang IC, Ackerson TJ, Major ML, Detrisac CJ, Kalinichenko VV, et al. Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice. Cancer Res. 2006;66:1712–20.PubMed CentralPubMedView ArticleGoogle Scholar
- Krupczak-Hollis K, Wang X, Kalinichenko VV, Gusarova GA, Wang IC, Dennewitz MB, et al. The mouse Forkhead Box m1 transcription factor is essential for hepatoblast mitosis and development of intrahepatic bile ducts and vessels during liver morphogenesis. Dev Biol. 2004;276:74–88.PubMedView ArticleGoogle Scholar
- Ye H, Holterman AX, Yoo KW, Franks RR, Costa RH. Premature expression of the winged helix transcription factor HFH-11B in regenerating mouse liver accelerates hepatocyte entry into S phase. Mol Cell Biol. 1999;19:8570–80.PubMed CentralPubMedGoogle Scholar
- Wonsey DR, Follettie MT. Loss of the forkhead transcription factor FoxM1 causes centrosome amplification and mitotic catastrophe. Cancer Res. 2005;65:5181–9.PubMedView ArticleGoogle Scholar
- Kalinichenko VV, Major ML, Wang X, Petrovic V, Kuechle J, Yoder HM, et al. Foxm1b transcription factor is essential for development of hepatocellular carcinomas and is negatively regulated by the p19ARF tumor suppressor. Genes Dev. 2004;18:830–50.PubMed CentralPubMedView ArticleGoogle Scholar
- Kim IM, Ackerson T, Ramakrishna S, Tretiakova M, Wang IC, Kalin TV, et al. The Forkhead Box m1 transcription factor stimulates the proliferation of tumor cells during development of lung cancer. Cancer Res. 2006;66:2153–61.PubMedView ArticleGoogle Scholar
- Liu M, Dai B, Kang SH, Ban K, Huang FJ, Lang FF, et al. FoxM1B is overexpressed in human glioblastomas and critically regulates the tumorigenicity of glioma cells. Cancer Res. 2006;66:3593–602.PubMedView ArticleGoogle Scholar
- Chan DW, Yu SY, Chiu PM, Yao KM, Liu VW, Cheung AN, et al. Over-expression of FOXM1 transcription factor is associated with cervical cancer progression and pathogenesis. J Pathol. 2008;215:245–52.PubMedView ArticleGoogle Scholar
- Li Q, Zhang N, Jia Z, Le X, Dai B, Wei D, et al. Critical role and regulation of transcription factor FoxM1 in human gastric cancer angiogenesis and progression. Cancer Res. 2009;69:3501–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Uddin S, Ahmed M, Hussain A, Abubaker J, Al-Sanea N, AbdulJabbar A, et al. Genome-wide expression analysis of Middle Eastern colorectal cancer reveals FOXM1 as a novel target for cancer therapy. Am J Pathol. 2011;178:537–47.PubMed CentralPubMedView ArticleGoogle Scholar
- Gemenetzidis E, Bose A, Riaz AM, Chaplin T, Young BD, Ali M, et al. FOXM1 upregulation is an early event in human squamous cell carcinoma and it is enhanced by nicotine during malignant transformation. PLoS One. 2009;4, e4849.PubMed CentralPubMedView ArticleGoogle Scholar
- Teh MT, Hutchison IL, Costea DE, Neppelberg E, Liavaag PG, Purdie K, et al. Exploiting FOXM1-orchestrated molecular network for early squamous cell carcinoma diagnosis and prognosis. Int J Cancer. 2013;132:2095–106.PubMedView ArticleGoogle Scholar
- Scheid MP, Woodgett JR. Protein kinases: six degrees of separation? Curr Biol. 2000;10:R191–4.PubMedView ArticleGoogle Scholar
- Rahman MA, Amin AR, Shin DM. Chemopreventive potential of natural compounds in head and neck cancer. Nutr Cancer. 2010;62:973–87.PubMed CentralPubMedView ArticleGoogle Scholar
- Marquette C, Nabell L. Chemotherapy-resistant metastatic breast cancer. Curr Treat Options Oncol. 2012;13:263–75.PubMedView ArticleGoogle Scholar
- Wang IC, Meliton L, Tretiakova M, Costa RH, Kalinichenko VV, Kalin TV. Transgenic expression of the forkhead box M1 transcription factor induces formation of lung tumors. Oncogene. 2008;27:4137–49.PubMedView ArticleGoogle Scholar
- Hegde NS, Sanders DA, Rodriguez R, Balasubramanian S. The transcription factor FOXM1 is a cellular target of the natural product thiostrepton. Nat Chem. 2011;3:725–31.PubMedView ArticleGoogle Scholar
- Bhat UG, Halasi M, Gartel AL. FoxM1 is a general target for proteasome inhibitors. PLoS One. 2009;4, e6593.PubMed CentralPubMedView ArticleGoogle Scholar
- Uddin S, Hussain AR, Ahmed M, Siddiqui K, Al-Dayel F, Bavi P, et al. Overexpression of FoxM1 offers a promising therapeutic target in diffuse large B-cell lymphoma. Haematologica. 2012;97:1092–100.PubMed CentralPubMedView ArticleGoogle Scholar
- Ahmed M, Uddin S, Hussain AR, Alyan A, Jehan Z, Al-Dayel F, et al. FoxM1 and its association with matrix metalloproteinases (MMP) signaling pathway in papillary thyroid carcinoma. J Clin Endocrinol Metab. 2012;97:E1–13.PubMedView ArticleGoogle Scholar
- Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984;22:27–55.PubMedView ArticleGoogle Scholar
- Antonsson B, Montessuit S, Sanchez B, Martinou JC. Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem. 2001;276:11615–23.PubMedView ArticleGoogle Scholar
- Hussain AR, Ahmed M, Al-Jomah NA, Khan AS, Manogaran P, Sultana M, et al. Curcumin suppresses constitutive activation of nuclear factor-kappa B and requires functional Bax to induce apoptosis in Burkitt's lymphoma cell lines. Mol Cancer Ther. 2008;7:3318–29.PubMedView ArticleGoogle Scholar
- Al-Maghrabi J, Buhmeida A, Emam E, Syrjanen K, Sibiany A, Al-Qahtani M, et al. Cyclooxygenase-2 expression as a predictor of outcome in colorectal carcinoma. World J Gastroenterol. 2012;18:1793–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Li D, Wei P, Peng Z, Huang C, Tang H, Jia Z, et al. The critical role of dysregulated FOXM1-PLAUR signaling in human colon cancer progression and metastasis. Clin Cancer Res. 2013;19:62–72.PubMed CentralPubMedView ArticleGoogle Scholar
- Seitz C, Hugle M, Cristofanon S, Tchoghandjian A, Fulda S. The dual PI3K/mTOR inhibitor NVP-BEZ235 and chloroquine synergize to trigger apoptosis via mitochondrial-lysosomal cross-talk. Int J Cancer. 2012;132(11):2682–93.PubMedView ArticleGoogle Scholar
- Peddaboina C, Jupiter D, Fletcher S, Yap JL, Rai A, Tobin R, et al. The downregulation of Mcl-1 via USP9X inhibition sensitizes solid tumors to Bcl-xl inhibition. BMC Cancer. 2012;12:541.PubMed CentralPubMedView ArticleGoogle Scholar
- Itatsu K, Sasaki M, Yamaguchi J, Ohira S, Ishikawa A, Ikeda H, et al. Cyclooxygenase-2 is involved in the up-regulation of matrix metalloproteinase-9 in cholangiocarcinoma induced by tumor necrosis factor-alpha. Am J Pathol. 2009;174:829–41.PubMed CentralPubMedView ArticleGoogle Scholar
- Bu X, Zhao C, Dai X. Involvement of COX-2/PGE (2) Pathway in the Upregulation of MMP-9 Expression in Pancreatic Cancer. Gastroenterol Res Pract. 2011;2011:214269.PubMed CentralPubMedView ArticleGoogle Scholar
- Bartholomeusz C, Gonzalez-Angulo AM. Targeting the PI3K signaling pathway in cancer therapy. Expert Opin Ther Targets. 2012;16:121–30.PubMedView ArticleGoogle Scholar
- Yao M, Lam EC, Kelly CR, Zhou W, Wolfe MM. Cyclooxygenase-2 selective inhibition with NS-398 suppresses proliferation and invasiveness and delays liver metastasis in colorectal cancer. Br J Cancer. 2004;90:712–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Bavi P, Prabhakaran SE, Abubaker J, Qadri Z, George T, Al-Sanea N, et al. Prognostic significance of TRAIL death receptors in Middle Eastern colorectal carcinomas and their correlation to oncogenic KRAS alterations. Mol Cancer. 2010;9:203.PubMed CentralPubMedView ArticleGoogle Scholar
- Bavi P, Jehan Z, Atizado V, Al-Dossari H, Al-Dayel F, Tulbah A, et al. Prevalence of fragile histidine triad expression in tumors from saudi arabia: a tissue microarray analysis. Cancer Epidemiol Biomarkers Prev. 2006;15:1708–18.PubMedView ArticleGoogle Scholar
- Camp RL, Dolled-Filhart M, Rimm DL. X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization. Clin Cancer Res. 2004;10:7252–9.PubMedView ArticleGoogle Scholar
- Kwok JM, Myatt SS, Marson CM, Coombes RC, Constantinidou D, Lam EW. Thiostrepton selectively targets breast cancer cells through inhibition of forkhead box M1 expression. Mol Cancer Ther. 2008;7:2022–32.PubMedView ArticleGoogle Scholar
- Hussain AR, Ahmed SO, Ahmed M, Khan OS, Al Abdulmohsen S, Platanias LC, et al. Cross-talk between NFkB and the PI3-kinase/AKT pathway can be targeted in primary effusion lymphoma (PEL) cell lines for efficient apoptosis. PLoS One. 2012;7, e39945.PubMed CentralPubMedView ArticleGoogle Scholar
- Hussain AR, Al-Rasheed M, Manogaran PS, Al-Hussein KA, Platanias LC, Al Kuraya K, et al. Curcumin induces apoptosis via inhibition of PI3'-kinase/AKT pathway in acute T cell leukemias. Apoptosis. 2006;11:245–54.PubMedView ArticleGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.