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Association of XPO1 Overexpression with NF-κB and Ki67 in Colorectal Cancer

Association of XPO1 Overexpression with NF-κB and Ki67 in Colorectal Cancer Objectives: Exportin 1(XPO1), a nuclear exporter protein, has been gaining recognition in cancer progression and treatment. This study aimed to evaluate the association between the overexpression of XPO1 with NF-κB, Ki67 and clinicopathological characteristics in colorectal cancer (CRC) tissue samples and to explore the anti-proliferative effect of KPT-330, as XPO1 inhibitor, in colorectal cancer cell line. Methods: Forty CRC tissue samples were analyzed by immunostaining for the expressions of XPO1, NF-κB and Ki67 and then the anti-proliferative effect of the KPT-330 was also evaluated in HT29 colorectal cancer cell line. Results: XPO1 overexpression was observed in 52.5% of CRC and significantly apparent with strong intensity in tumor cells compared to the normal adjacent epithelium (P<0.001). Regarding to the histopathological characteristics, the XPO1 overexpression significantly associated with advanced tumor stages (P=0.049) and has great tendency towards moderate/poorly differentiated tumors. Although the XPO1 overexpression was strongly associated with high Ki67 expression (P=0.001), only Ki67 expression showed significant association with tumor size (P=0.012). No significant association was detected between the XPO1 overexpression and NF-κB, while the NF-κB positive expression was significantly associated with lymph node metastasis and Ki67 expression at P=0.027 and P= 0.007, respectively. The in vitro experiments showed a great impact of KPT-330, as XPO1 inhibitor, to inhibit cancer growth in dose and time dependent manner and significantly diminished the colony formation (P<0.001) of HT29 cells- associated with the expression of Ki67 (P<0.001). Conclusion: XPO1 overexpression and NF-κB expression may serve as potential biomarker associated with CRC pathogenesis and proliferation, while the KPT-330 is effectively inhibited-colon cancer growth in vitro. Further studies considering the prognostication role of XPO1 overexpression in CRC are required. Keywords: Colorectal cancer (CRC)- Exportin 1 (XPO1)- NF-κB- Ki67- KPT-330 Asian Pac J Cancer Prev, 20 (12), 3747-3754 Kashyap et al., 2016). In cancer, the disruption of XPO1 Introduction activity may be involved in the increasing of survival and proliferative rate activity of tumor cells (Turner and Colorectal cancer (CRC) is one of the most common Sullivan, 2008). Preclinical data and many clinical trials cancers, implicated as the third causative cancer morbidity used the developed new generation of selective inhibitor and the fourth of cancer mortality worldwide (Torre of nuclear export (SINE) compounds as XPO1 inhibitor, et al., 2015). Despite recent advances in therapeutic such as KPT-330, has a property of low toxicity and good approaches that have considerable practical value, the patients’ tolerability as it was seen in both hematological prognosis of more than 50% of CRC patients still remains malignancies and solid tumors. This provided an excellent poor (Bai et al., 2015). The uncontrolled expansion of model in translational medicine for targeting of XPO1 proliferative activity propelled by the disruption of cellular activity in cancer therapy with promising efficacy in recent homeostasis may play a major role to increase the growth years (Abdul Razak et al., 2016). and survival rates of CRC tumor cells (Sancho et al., Association of XPO1 overexpression with high tumor 2004). Exportin 1 (XPO1), also known as chromosome grade and advanced tumor stage (Noske et al., 2008; Shen region maintenance 1 (CRM1) protein, is involved in the et al., 2009) as well as with poor prognosis was noted in homeostatic of nucleocytoplasmic transport of over 200 some tumor cancers such as gastric (Zhou et al., 2013), known cargos, most of them are tumor suppressor and ovarian (Noske et al., 2008) and acute leukemia (Kojima cell cycle regulatory proteins such as p53, pRb, FOXOs, et al., 2013), but little is known about such association BRCA1/2 and inhibitor of NF-κB (Kau et al., 2004; 1 3 Department of Pathobiology, Faculty of Science, Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Department of Pathology, Faculty of Medicine and Health Sciences, University of Sana’a, Sana’a, Yemen. *For Correspondence: [email protected] Asian Pacific Journal of Cancer Prevention, Vol 20 3747 Mohammed Aladhraei et al between the XPO1 overexpression and clinicopathological (ATCC). The HT29 cell was maintained in DMEM/F12 characteristics in CRC. In colon cancer cell lines, the supplemented with 10% fetal bovine serum (FBS) (Sigma) XPO1 increases the oncogenic activity of tumor cells and was cultured at 37°C in a humidified incubator of through mislocalization of some proteins such as p27 and 5% CO . XPO1 inhibitor (KPT-330) was obtained from survivin (Ferreiro-Neira et al., 2016; Heong et al., 2016). Karyopharm Therapeutics. DMSO was used as diluent On the other hand, In CRC, NF-κB over-expression was control for all in vitro studies. considered as biomarker associated with worse 3 and 5 years overall survival (Wu et al., 2015). Therefore, Immunohistochemical analysis exploration, in to what extent, the involvement of XPO1 Tissue sections from FFPE tissue blocks with 4μm overexpression in CRC characteristics in tissue samples, thickness were cut and mounted on positive charge glass considering the histopathological characteristics and slides and processed by standard procedures for IHC proliferation activity, in association with the NF-κB in parallel with positive and negative controls for each may be helpful in line of individualizing of patients antibody as described previously (Noske et al., 2008). treatment using XPO1 inhibitor. Accordingly, in the Endogenous peroxidase activity was blocked using 3% present study, we tried to address the differences hydrogen peroxide (H O ) and antigen retrieval was 2 2 between the overexpression of XPO1 in CRC tumor done in citrate buffer (pH 6.0) using microwave oven. cells and adjacent normal epithelium. Then we explored Then, the sections were incubated with monoclonal the association between the XPO1 overexpression and primary antibodies against XPO1 (1:400), NF-κB clinico-histopathological features as well as with NF-κB (1:200) and Ki-67 (1:500) overnight at 4°C, followed and proliferative marker, Ki67, in CRC tissue samples. by a detection system HiDef Detection™ Amplifier and Furthermore, we demonstrated the anti-proliferative then a HRP Polymer Detector (anti-mouse/rabbit, Cell effect of XPO1 inhibitor, KPT-330, on HT29 colrectal Marque, USA) for 10 minutes in each step. The DAB cancer cell line. chromogen (3,3-diaminobenzidine tetrahydrochloride) then added with substrate for 5 minutes and the sections were washed in distilled water and then counterstained Materials and Methods with hematoxylin for 2 minutes. The tissue sections were Patients and clinicopathological data rinsed with PBS (pH 7.3) between each step during IHC Samples in the form of formalin fixed paraffin processing. embedded (FFPE) tissue blocks were collected randomly from 40 patients who underwent surgical resection and IHC evaluation of XPO1, NF-κB and Ki67 expression diagnosed as colorectal carcinoma between December Two independent investigators evaluated each 2016 and October 2017 at National Oncology Center section. The intensity scoring and positive staining (NOC), Sana’a, Republic of Yemen. Age and sex of the cells were used to define the XPO1 expression in CRC patients as well as tumor size were retrieved from the tumor cells and adjacent normal epithelium. The XPO1 histopathological reports. The degree of histological expression was calculated by using the intensity score differentiation of tumors were categorized according to multiplied with positive cells score. The intensity of percentage of the gland-like structures formation and XPO1 immunostaining scored as 0 (negative), 1 (weak), grouped in to well and moderately/poorly differentiation 2 (moderate), or 3 (strong) for either the nuclear, nuclear (Xiao et al., 2013). The lymph node metastasis was membranous and/or cytoplasmic area. The localization also categorized in to negative (no regional lymph node staining that diagnosed with positive cells scored according metastasis) and positive (metastasis in to regional lymph to the percentage of immunoreactive cells as 0 (none), 1 nodes). The tumor stages were classified according to the (<10%), 2 (10–50%) and 3 (>50%) and multiplied by the TNM staging system, the American Joint Committee of intensity score to give 0 score (negative expression), 1-3 Cancer (AJCC), and grouped in to stage I-II and stage (weak expression), 4-6 (moderate expression) and 7-9 III-IV. Hematoxylin and eosin (H&E) stained sections (strong expression). The XPO1 expression levels were were reviewed to confirm the presence of >50% of tumor categorized in to non-overexpression (negative, weak cells with adjacent normal epithelial for further study with or moderate expressions) and over-expression (strong immunohistochemistry (IHC) staining. Ethical approval expression) (Gravina et al., 2015). for this study was obtained from National Health and The evaluation of NF-κB (p65) was done according Medical Research Committee (NHMRC), Republic of the percentage of immunoreactive cells (quantity score) Yemen (B2/10-2017) and the informed consent was taken with the staining intensity in 10 high power visual fields of from all the CRC patients. tumor cells. The percentage of immunoreactive tumor cells was evaluated as follow: no staining as 0, 1%-10% of cells Antibodies and Reagents stained as 1, 11%-50% cells stained as 2, 51%-80% cells The anti-XPO1 antibody was obtained from Santa stained as 3, and 81%-100% cells stained as 4. Staining Cruz Biotechnology, XPO1/CRM1 (sc-74454), and the intensity was rated on a scale of 0-3, with 0 = negative, 1 monoclonal anti-NF-κB p65 antibody (phosphor S536) = weak, 2 = moderate, and 3= strong. The IHC staining purchased from abcam, USA, while the anti-Ki67 antibody of NF-κB was considered positive if the multiplied scored (MIB-1) was obtained from Cell Marque, USA. For in >3/12 (Long et al., 2008). The Ki67 expression was vitro experiments, colorectal cancer cell line HT29 was assessed in 10 representative high power visual fields of obtained from the American Type Culture Collection tumor cells with cutoff value of <40% considered as low 3748 Asian Pacific Journal of Cancer Prevention, Vol 20 DOI:10.31557/APJCP.2019.20.12.3747 XPO1 Overexpression with NF-κB and Ki67 in CRC and ≥40% as high nuclear Ki67 expression (Salminen et for further 48 hours. The cells then washed with PBS, al., 2005). fixed in cold acetone immersed in hydrogen peroxide for 10 minutes. After that, the cells incubated with anti-Ki67 Cell growth inhibition assay (1:1,000) antibody for one hour followed by incubation in Cells were seeded at density 10 cells/well in 96-well the HiDef Detection™ Amplifier and HiDef Detection™ plates, then the cells were treated next day with series HRP Polymer Detector for 10 minutes in each. The cells doses below and above their IC concentrations of then incubated with DAB-chromogen system for 3-5 KPT-330, 0.25, 0.5, 1, and 2μm/L and then further minutes followed by counterstain in hematoxylin. Scoring incubated for 24, 48 and 72 hours respectively. Thereafter, of Ki67 was done depending on the percentage of positive the cells were subjected to cell proliferation analysis in cells (0% - 100%) of the total cells numbers in high-power consecutive days using MTT [3-(4,5-dimethylthiazol-2- fields, as in previous study (Zu et al., 2012). yl)-2,5-diphenyltetrazolium bromide] assay and incubated for 4 hours. After that, the cells were lysed by 200 µL Statistical analysis of DMSO. Then the spectrophotometric absorbance of In this study, all analysis was performed using the samples was determined using a microplate reader SPSS version 18 statistical software program (SPSS (Bio-Rad) (Ferreiro-Neira et al., 2016). Inc., Chicago, IL, USA). Chi-square and Fisher ’s exact tests were used to find the association between Colony formation assay the overexpression of XPO1, NF-κB and Ki67 with The ability of KPT-330 to inhibit the HT29 cell clinico-histopathological factures. Mann-Whitney U lines colonies formation was assessed by the colony test was used with continuous variables. All cell culture formation assay as described previously (Niu et al., experiments were performed in triplicate and repeated at 2015). Briefly, 10 HT29 cells were seeded into 24-well least three times while the one-way ANOVA was used to plates in triplicates and then treated with vehicle control compare the mean between groups. The P values <0.05 (DMSO) and series doses of KPT-330, 0.5, 1, 2µmol/L) was considered statistically significant difference. for 24 hours. The culture medium was changed and the cells were incubated to allow colonies formation. After Results two weeks, cells were fixed with 5% glutaraldehyde in PBS and stained with 0.1% crystal violet. The numbers Expressions of XPO1, NF-κB and Ki67 in CRC of colonies formed were confirmed by manual counting The average age of the patients was 49.9 years (range for each dose. 24-80), with 55% (22/40) were females. After we used the IHC analysis, the majority of XPO1 expressions were Immunocytochemical (ICC) expressions of Ki67 in HT29- nuclear and/or nuclear membrane with weak to moderate KPT-330-treated cells diffuse cytoplasmic staining. The XPO1 expression was The HT29 cells seeded on cover slips in 6-well identified and scored as negative (15%), weak (12.5%), at density of 10 cells, incubated overnight and then moderate (20%) and strong (52.5%). The only strong score subjected to increasing doses of KPT-330 and incubated was considered as XPO1 overexpression (52.5%) while Figure 1. XPO1 Expression in Adjacent Normal Epithelium (A1 and B) and Tumor Cells (A2 and C). (Original Magnification- 200x and 400x, respectively). Asian Pacific Journal of Cancer Prevention, Vol 20 3749 Mohammed Aladhraei et al Figure 2. Association of XPO1 Overexpression and NF-κB with Ki67. The IHC expressions of both XPO1 (A) and NF-κB (B) show highly concordance of immunoreactivity within the tissue sections expressed Ki67 (C and D respectively) (original magnification- 400x). the 47.5% were considered as XPO1 non-overexpression. Although there was no big differences in the frequency of the XPO1 immunostaining between the tumor cells and normal adjacent epithelium, the intensity of XPO1 expression was abundant, intense and more apparent in tumor cells compared to the adjacent normal epithelial with significant difference (P<0.001) (Table 1 and Figure 1). The positive NF-κB expression was noted in 32.5% (13/40) while the Ki67 was observed as high expression in 72.5% (29/40) of CRC tumors (Table 2). Association between the XPO1 overexpression and NF-κB, Ki67 with clinicopathological features of CRC tissue samples In this study we identified a high tendency of XPO1 overexpression in moderately/poorly differentiated tumors and was significantly associated with advanced tumor stage (III/IV) (P=0.059 and P=0.049, respectively) (Table 2). Although the XPO1 overexpression was frequently noted in tumors with increasing numbers of positive lymph nodes metastasis, this tendency did not reach to the statistical significance difference (P=0.308). Significantly, we found a strong concordance between the XPO1 overexpression and high Ki67 expression in the tumor cells within the most tissue sections that showed immunoreactivity for both XPO1 and Ki67, and the XPO1 Table 1. Expression of XPO1 in CRC Tumors Cells and Adjacent Normal Epithelium XPO1 Number of patients (%) P value Figure 3. Correlation between the XPO1 overexpression expression Normal (%) Cancer (%) and Ki67 with tumor size. Mann-Whitney test demonstrate Negative 3 (7.5) 6 (15.0) that the tumors with larger size exhibit significant high Ki67 expression (A) (P=0.012) and to some extent the Weak 18 (45.0) 5 (12.5) XPO1 overexpression (B) (P=0.168). * represents the Moderate 15 (37.5) 8 (20.0) significant statistical differences between groups at P<0.05. Strong 4 (10.0) 21 (52.5) <0.001 3750 Asian Pacific Journal of Cancer Prevention, Vol 20 DOI:10.31557/APJCP.2019.20.12.3747 XPO1 Overexpression with NF-κB and Ki67 in CRC Figure 4. KPT-330 Inhibits the Growth and Proliferation of HT29 Cell Line. MTT assay (A), note the decreases of rate cell growth after 24, 48 and 72 hours of incubation while the colony formation assay (B) evince the ability of KPT-300 to inhibit the colonies formation. The ICC (C) staining shows reversible results of cell-expressed-ki67 and KPT-330 at doses 0.5 to 2µmol/L. *,** represent the statistical differences between the treated and untreated cells (P<0.001 and P<0.0001, respectively). overexpression was strongly associated with the high Ki67 the high Ki67 expression (P=0.007) (Table 2 and Figure 2) expression (P=0.001) (Table 2 and Figure 2). On the same manner, the CRC tumors sizes in this study ranges from KPT-330 inhibits cell proliferation, colony formation and 3 to 11 cm with mean 6.6 cm and we noted that the high Ki67 expression of HT29 cells nuclear Ki67 expression was associated with an increase As shown at Figure 4A, the increased of KPT-330 serial tumor size (P=0.012) but not XPO1 (P=0.168), with concentrations from 0.25, 0.5 and 1 to 2 µmol/L induced Mann-Whitney test, as it is illustrated in Figure 3. In this the cells growth inhibition after 72 hours of incubation. study, although the XPO1 overexpression was noticed in The IC value was 0.9 µmol/L in HT29 cells. These results 8/13 of positive NF-κB expression tumors, the statistical revealed that, when the KPT-330 dose exceeds 1 µmol/L significant association between the XPO1 overexpression and the action of time was >24 hours, the growth of cells and NF-κB was not found (P=0.427) (Table 3). However, was inhibited in a time and dose-dependent manner. most of the NF-κB positive tumors (11/13) were involved Further evaluation of the long-term effect of KPT-330 with positive lymph nodes metastasis with significant on HT29 growth, the clonogenic assay was performed difference (P=0.027) and showed strong association with for 2 weeks. The results revealed that the colonies Table 2. Association of XPO1 Overexpression and NF-κB with Clinicopathological Features and Ki67 Expression Clinicopathological Patients XPO1 expression P-value NF-κB expression P-value features and Ki67 expression No (%) Nonoverex-pressio Overexpr-ession -ve +ve Tumor differentiation 0.059 0.906 Well 19 (47.5) 12 7 13 6 Moderate/Poor 21 (52.5) 7 14 14 7 Lymph node metastasis 0.366 0.027* Negative 16 (40) 9 7 14 2 Positive 24 (60) 10 14 13 11 Tumor stage 0.049* 0.286 I–II 11 (27.5) 8 3 4 7 III-IV 29 (72.5) 11 18 7 22 Ki67 0.001** 0.007** <40% 11 (27.5) 10 1 11 0 ≥40% 29 (72.5) 9 20 16 13 *,** represent the statistical P<0.001 and P<0.0001, respectively. Asian Pacific Journal of Cancer Prevention, Vol 20 3751 Mohammed Aladhraei et al CRC tissues which may attribute to the large differences in Table 3. Association between the XPO1 Overexpression and NF-κB in CRC the sample sizes in each of well and poorly differentiated tumor groups; others were found such association in other NF-κB Patients XPO1 expression P value types of cancers such as ovarian cancer (Noske et al., expression No (%) Nonoverex- Overexpr- 2008), glioma (Shen et al., 2009) and the XPO1 expression pressio ession was increased from well to poorly differentiated breast Negative 27 (67.5) 14 13 tumors (Yue et al., 2018). Additionally, we noted that the Positive 13 (32.5) 5 8 0.427 poorly differentiated tumors were significantly increased numbers of metastatic lymph nodes, which goes in line formation decreased significantly by 86%, 52% and 21% with a cohort study results of 124,180 CRC patients by in response to KPT-330 doses of 0.5, 1 and 2 μmol/L, Ricciardi et al., (2006) who concluded that the poorly respectively (Figure 4B). Moreover, the Ki67 ICC differentiated tumors were much more likely to be with staining of KPT-330-treated-HT29 cells showed that the lymph node positive than well-differentiated tumors. Ki67 positive cells in the KPT-330 treated groups was In this study, most of moderately/ poorly differentiated significantly reduce compared to the untreated (Figure tumors with XPO1 overexpression were involved by 4C). These results were consistent with the MTT and lymph node metastasis. Moreover, the clinical trial colony formation assays results, confirming that the carried by Mau-Soerensen et al., (2014) among advanced KPT-330 inhibits the proliferation of HT29 colon cancer metastatic CRC patients and used oral KPT-330 for 28 cell line in dose and time dependent manner. days confirmed the valuable of XPO1 inhibitors in line of disease stability for advanced staged CRC patients. Collectively, these results may add insights of XPO1 Discussion involvement in the CRC pathogenesis and progression XPO1 is the major mammalian exporter protein that leading to poorly differentiated tumors and advance tumor facilitates the nucleocytoplasmic transport of over 200 stages. In some tumors such as lung cancer and mantle cell tumor suppressors and cell cycle regulatory proteins. Its lymphoma cells, the XPO1 inhibitors modulate the NF- overexpression reported in several types of tumors and was κB activity through trapping of IκB in the nucleus, which correlated with aggressive behavior and poor survival (Sun is target of XPO1 for nucleocytoplasmic transportation, et al., 2016). Furthermore, in recent years the XPO1 gains a leading to repression of NF-κB activity over time which great attention after launching a next-generation selinexor contributes in the growth suppression and apoptosis (KPT-330) as selective inhibition of nuclear export of induction (Zhang et al., 2013). In this study we found that XPO1 with good properties regarding low toxicity in the XPO1 overexpression was associated, to some extent, vivo and good tolerability by the patients (Lapalombella with the NF-κB positive expression without significant et al., 2012; Hing et al., 2016). In CRC, little is known difference. On the other hand, it has been proved that about the overexpression of XPO1 in tissue samples and the activated NF-κB contributes in the progression of its association with histopathological features, NF-κB CRC through upregulation expression of diverse target and Ki67. Our previous study concerned of p53, reported genes that are involved in inflammation (cytokines), cell that the XPO1 positivity was associated with loss of p53 proliferation (Cyclin D1), angiogenesis (VEGF, IL-8, expression in CRC tumors with lymph node metastasis COX2), and metastasis (MMP9) (Wang et al., 2009; Xie (Aladhraei et al., 2019). In the current study, we noticed et al., 2019) make it an interesting tumor marker in CRC a significant apparent of XPO1 overexpression in CRC pathogenesis. In this regard, the results of our study showed tumor cells compared to the adjacent normal epithelium that the NF-κB expression was significantly associated as it was reported in many other types of cancers such as with positive lymph node metastasis. Furthermore, strong esophageal (van der Watt et al., 2014), gastric (Subhash et significant association between the expressions of NF-κB al., 2018), lung (Gao et al., 2015), and ovarian (Noske et and high Ki67 expression was identified. These findings al., 2008) cancers, as well as leukemic cells (Kojima et al., were consistent with in vitro studies (Lu et al., 2016) as 2013). The overexpression of XPO1 within the tumor cells well as with NF-κB activity role in CRC tumors (Meteoglu may reflect its abundance and suggests a gain-of-function et al., 2015). These results add an insight in line of NF-κB or oncogenic activity in line of further CRC pathogenesis involvement in CRC metastasis and proliferation makes a (Conforti et al., 2015). Crochiere et al., (2016) suggested rational for targeting of NF-κB in CRC as it was reported that the abundance of XPO1 within the tumor cells might (Sakamoto and Maeda, 2010). It is well known that the be able to predict the drug resistance. sustained proliferation is one of the cancer hallmarks As it is known, the tumor grade is considered as acquired during cancer development and progression stage-independent prognostic factor in CRC, and the (Hanahan and Weinberg, 2011). In this regard, we poorly differentiated tumors are associated with poor identified that the overexpression of XPO1 was strongly patient survival (Fleming, et al., 2012). In this study associated with Ki67 expression, which in turn reflected we identified a high tendency of XPO1 overexpression the implication of XPO1 overexpression in mislocalization towards the moderate/poorly differentiated tumors as well of essential cell cycle inhibitory proteins such as p27, as increase of XPO1 overexpression frequency in advance p53, cyclins and some apoptotic proteins (Nguyen et al., tumor stages III-IV with significant difference. Although 2012; Niu et al., 2015) lead to unregulated cell division Shintani et al., (2016) studied the XPO1 expression in and increased tumor size. Despite ascending tendency CRC tissue samples, they did not find such association in of XPO1 expression was noticed with increased tumor 3752 Asian Pacific Journal of Cancer Prevention, Vol 20 DOI:10.31557/APJCP.2019.20.12.3747 XPO1 Overexpression with NF-κB and Ki67 in CRC size, our study did not find a direct association between XPO1 overexpression prognostication in CRC patients the XPO1 overexpression and tumor size; while the high may be recommended. Ki67 expression was significantly noticed in larger tumor sizes. The strong association between the overexpression Acknowledgements of XPO1 and high Ki67 expression in CRC patients’ tumors was in concordance with our in vitro results that The authors are grateful to the Department of confirmed the anti-proliferative effect of KPT-330. The Pathobiology, Faculty of Science, Mahidol University KPT-330 induced the growth inhibition and suppressed for the providing the laboratory space and facilities as the HT29 colorectal cancer cell line proliferation in dose well as the excellent technical assistance assisted by and time dependent manner as shown by the MTT assay. Mrs. Pranom Puchadapirom and Mrs. Maliwan Emyeam. Furthermore, the experiments showed a great stability of Ethically, the study protocol followed the guidelines KPT-330 in vitro, which means a continuous inhibition approved by the Ethical Committee of the Medical of growth over the time during incubation and persisted Research of the National Health and Medical Research for up to 72 hours. This explains the powerful binding of Committee (NHMRC), Republic of Yemen (Approval No. KPT-330 in slowly reversible action with XPO1 leading B2/10-2017). No potential conflict of interest relevant to to inhibition the binding between the cargoes proteins this article was reported. and XPO1 in HT29 cancer cell line which in turn induce nuclear retention of cell cycle regulatory proteins, inhibits References the proliferation and may initiate the apoptosis (Draetta et Abdul Razak AR, Mau-Soerensen M, Gabrail NY, et al (2016). al., 2011; Senapedis et al., 2014). The long-term effect of First-in-class, first-in-human phase i study of selinexor, KPT-330 on the HT29 colony formation was significantly a selective inhibitor of nuclear export, in patients with seen as a decrease of colonies formation with increase advanced solid tumors. J Clin Oncol, 34, 4142-50. of KPT-330 concentration. These findings were further Aladhraei M, Al-Salami E, Poungvarin N, Suwannalert P (2019). supported by the evaluation of Ki67 expression by ICC in The roles of p53 and XPO1 on colorectal cancer progression HT29-KPT-330-treated cells. The number of HT29 cells in Yemeni patients. J Gastrointest Oncol, 10, 437-44. with Ki67 expression was dramatically and significantly Azmi AS, Li Y, Muqbil I, et al (2017). Exportin 1 (XPO1) decreased in HT29 KPT-330-treated groups compared inhibition leads to restoration of tumor suppressor miR-145 to the control, and this confirmed the anti-proliferative and consequent suppression of pancreatic cancer cell proliferation and migration. Oncotarget, 8, 82144-55. effect of KPT-330. In fact, the inhibition of XPO1 activity Bai W, Wu Y, Zhang P, Xi Y (2015). Correlations between induced G1 cell cycle arrest with the loss of S, G2, and M expression levels of thymidylate synthase, thymidine phases within hours of application followed by increase phosphorylase and dihydropyrimidine dehydrogenase, and of nuclear cell cycle inhibitory proteins such as p27 and efficacy of 5-fluorouracil-based chemotherapy for advanced p53 (Niu et al., 2015). Normally, the Ki67 is degraded colorectal cancer. 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Annu Rev Cell Dev Biol, Non Commercial 4.0 International License. 20, 695-723. Senapedis WT, Baloglu E, Landesman Y (2014). Clinical translation of nuclear export inhibitors in cancer. Semin Cancer Biol, 27, 74-86. Shen A, Wang Y, Zhao Y,et al (2009). Expression of CRM1 in 3754 Asian Pacific Journal of Cancer Prevention, Vol 20 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Asian Pacific Journal of Cancer Prevention Pubmed Central

Association of XPO1 Overexpression with NF-κB and Ki67 in Colorectal Cancer

Aladhraei, Mohammed; Al-Thobhani, Abdulla Kassem; Poungvarin, Naravat; Suwannalert, Prasit
Asian Pacific Journal of Cancer Prevention , Volume 20 (12) – Dec 1, 2019
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Pubmed Central
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1513-7368
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2476-762X
DOI
10.31557/APJCP.2019.20.12.3747
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Abstract

Objectives: Exportin 1(XPO1), a nuclear exporter protein, has been gaining recognition in cancer progression and treatment. This study aimed to evaluate the association between the overexpression of XPO1 with NF-κB, Ki67 and clinicopathological characteristics in colorectal cancer (CRC) tissue samples and to explore the anti-proliferative effect of KPT-330, as XPO1 inhibitor, in colorectal cancer cell line. Methods: Forty CRC tissue samples were analyzed by immunostaining for the expressions of XPO1, NF-κB and Ki67 and then the anti-proliferative effect of the KPT-330 was also evaluated in HT29 colorectal cancer cell line. Results: XPO1 overexpression was observed in 52.5% of CRC and significantly apparent with strong intensity in tumor cells compared to the normal adjacent epithelium (P<0.001). Regarding to the histopathological characteristics, the XPO1 overexpression significantly associated with advanced tumor stages (P=0.049) and has great tendency towards moderate/poorly differentiated tumors. Although the XPO1 overexpression was strongly associated with high Ki67 expression (P=0.001), only Ki67 expression showed significant association with tumor size (P=0.012). No significant association was detected between the XPO1 overexpression and NF-κB, while the NF-κB positive expression was significantly associated with lymph node metastasis and Ki67 expression at P=0.027 and P= 0.007, respectively. The in vitro experiments showed a great impact of KPT-330, as XPO1 inhibitor, to inhibit cancer growth in dose and time dependent manner and significantly diminished the colony formation (P<0.001) of HT29 cells- associated with the expression of Ki67 (P<0.001). Conclusion: XPO1 overexpression and NF-κB expression may serve as potential biomarker associated with CRC pathogenesis and proliferation, while the KPT-330 is effectively inhibited-colon cancer growth in vitro. Further studies considering the prognostication role of XPO1 overexpression in CRC are required. Keywords: Colorectal cancer (CRC)- Exportin 1 (XPO1)- NF-κB- Ki67- KPT-330 Asian Pac J Cancer Prev, 20 (12), 3747-3754 Kashyap et al., 2016). In cancer, the disruption of XPO1 Introduction activity may be involved in the increasing of survival and proliferative rate activity of tumor cells (Turner and Colorectal cancer (CRC) is one of the most common Sullivan, 2008). Preclinical data and many clinical trials cancers, implicated as the third causative cancer morbidity used the developed new generation of selective inhibitor and the fourth of cancer mortality worldwide (Torre of nuclear export (SINE) compounds as XPO1 inhibitor, et al., 2015). Despite recent advances in therapeutic such as KPT-330, has a property of low toxicity and good approaches that have considerable practical value, the patients’ tolerability as it was seen in both hematological prognosis of more than 50% of CRC patients still remains malignancies and solid tumors. This provided an excellent poor (Bai et al., 2015). The uncontrolled expansion of model in translational medicine for targeting of XPO1 proliferative activity propelled by the disruption of cellular activity in cancer therapy with promising efficacy in recent homeostasis may play a major role to increase the growth years (Abdul Razak et al., 2016). and survival rates of CRC tumor cells (Sancho et al., Association of XPO1 overexpression with high tumor 2004). Exportin 1 (XPO1), also known as chromosome grade and advanced tumor stage (Noske et al., 2008; Shen region maintenance 1 (CRM1) protein, is involved in the et al., 2009) as well as with poor prognosis was noted in homeostatic of nucleocytoplasmic transport of over 200 some tumor cancers such as gastric (Zhou et al., 2013), known cargos, most of them are tumor suppressor and ovarian (Noske et al., 2008) and acute leukemia (Kojima cell cycle regulatory proteins such as p53, pRb, FOXOs, et al., 2013), but little is known about such association BRCA1/2 and inhibitor of NF-κB (Kau et al., 2004; 1 3 Department of Pathobiology, Faculty of Science, Clinical Molecular Pathology Laboratory, Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Department of Pathology, Faculty of Medicine and Health Sciences, University of Sana’a, Sana’a, Yemen. *For Correspondence: [email protected] Asian Pacific Journal of Cancer Prevention, Vol 20 3747 Mohammed Aladhraei et al between the XPO1 overexpression and clinicopathological (ATCC). The HT29 cell was maintained in DMEM/F12 characteristics in CRC. In colon cancer cell lines, the supplemented with 10% fetal bovine serum (FBS) (Sigma) XPO1 increases the oncogenic activity of tumor cells and was cultured at 37°C in a humidified incubator of through mislocalization of some proteins such as p27 and 5% CO . XPO1 inhibitor (KPT-330) was obtained from survivin (Ferreiro-Neira et al., 2016; Heong et al., 2016). Karyopharm Therapeutics. DMSO was used as diluent On the other hand, In CRC, NF-κB over-expression was control for all in vitro studies. considered as biomarker associated with worse 3 and 5 years overall survival (Wu et al., 2015). Therefore, Immunohistochemical analysis exploration, in to what extent, the involvement of XPO1 Tissue sections from FFPE tissue blocks with 4μm overexpression in CRC characteristics in tissue samples, thickness were cut and mounted on positive charge glass considering the histopathological characteristics and slides and processed by standard procedures for IHC proliferation activity, in association with the NF-κB in parallel with positive and negative controls for each may be helpful in line of individualizing of patients antibody as described previously (Noske et al., 2008). treatment using XPO1 inhibitor. Accordingly, in the Endogenous peroxidase activity was blocked using 3% present study, we tried to address the differences hydrogen peroxide (H O ) and antigen retrieval was 2 2 between the overexpression of XPO1 in CRC tumor done in citrate buffer (pH 6.0) using microwave oven. cells and adjacent normal epithelium. Then we explored Then, the sections were incubated with monoclonal the association between the XPO1 overexpression and primary antibodies against XPO1 (1:400), NF-κB clinico-histopathological features as well as with NF-κB (1:200) and Ki-67 (1:500) overnight at 4°C, followed and proliferative marker, Ki67, in CRC tissue samples. by a detection system HiDef Detection™ Amplifier and Furthermore, we demonstrated the anti-proliferative then a HRP Polymer Detector (anti-mouse/rabbit, Cell effect of XPO1 inhibitor, KPT-330, on HT29 colrectal Marque, USA) for 10 minutes in each step. The DAB cancer cell line. chromogen (3,3-diaminobenzidine tetrahydrochloride) then added with substrate for 5 minutes and the sections were washed in distilled water and then counterstained Materials and Methods with hematoxylin for 2 minutes. The tissue sections were Patients and clinicopathological data rinsed with PBS (pH 7.3) between each step during IHC Samples in the form of formalin fixed paraffin processing. embedded (FFPE) tissue blocks were collected randomly from 40 patients who underwent surgical resection and IHC evaluation of XPO1, NF-κB and Ki67 expression diagnosed as colorectal carcinoma between December Two independent investigators evaluated each 2016 and October 2017 at National Oncology Center section. The intensity scoring and positive staining (NOC), Sana’a, Republic of Yemen. Age and sex of the cells were used to define the XPO1 expression in CRC patients as well as tumor size were retrieved from the tumor cells and adjacent normal epithelium. The XPO1 histopathological reports. The degree of histological expression was calculated by using the intensity score differentiation of tumors were categorized according to multiplied with positive cells score. The intensity of percentage of the gland-like structures formation and XPO1 immunostaining scored as 0 (negative), 1 (weak), grouped in to well and moderately/poorly differentiation 2 (moderate), or 3 (strong) for either the nuclear, nuclear (Xiao et al., 2013). The lymph node metastasis was membranous and/or cytoplasmic area. The localization also categorized in to negative (no regional lymph node staining that diagnosed with positive cells scored according metastasis) and positive (metastasis in to regional lymph to the percentage of immunoreactive cells as 0 (none), 1 nodes). The tumor stages were classified according to the (<10%), 2 (10–50%) and 3 (>50%) and multiplied by the TNM staging system, the American Joint Committee of intensity score to give 0 score (negative expression), 1-3 Cancer (AJCC), and grouped in to stage I-II and stage (weak expression), 4-6 (moderate expression) and 7-9 III-IV. Hematoxylin and eosin (H&E) stained sections (strong expression). The XPO1 expression levels were were reviewed to confirm the presence of >50% of tumor categorized in to non-overexpression (negative, weak cells with adjacent normal epithelial for further study with or moderate expressions) and over-expression (strong immunohistochemistry (IHC) staining. Ethical approval expression) (Gravina et al., 2015). for this study was obtained from National Health and The evaluation of NF-κB (p65) was done according Medical Research Committee (NHMRC), Republic of the percentage of immunoreactive cells (quantity score) Yemen (B2/10-2017) and the informed consent was taken with the staining intensity in 10 high power visual fields of from all the CRC patients. tumor cells. The percentage of immunoreactive tumor cells was evaluated as follow: no staining as 0, 1%-10% of cells Antibodies and Reagents stained as 1, 11%-50% cells stained as 2, 51%-80% cells The anti-XPO1 antibody was obtained from Santa stained as 3, and 81%-100% cells stained as 4. Staining Cruz Biotechnology, XPO1/CRM1 (sc-74454), and the intensity was rated on a scale of 0-3, with 0 = negative, 1 monoclonal anti-NF-κB p65 antibody (phosphor S536) = weak, 2 = moderate, and 3= strong. The IHC staining purchased from abcam, USA, while the anti-Ki67 antibody of NF-κB was considered positive if the multiplied scored (MIB-1) was obtained from Cell Marque, USA. For in >3/12 (Long et al., 2008). The Ki67 expression was vitro experiments, colorectal cancer cell line HT29 was assessed in 10 representative high power visual fields of obtained from the American Type Culture Collection tumor cells with cutoff value of <40% considered as low 3748 Asian Pacific Journal of Cancer Prevention, Vol 20 DOI:10.31557/APJCP.2019.20.12.3747 XPO1 Overexpression with NF-κB and Ki67 in CRC and ≥40% as high nuclear Ki67 expression (Salminen et for further 48 hours. The cells then washed with PBS, al., 2005). fixed in cold acetone immersed in hydrogen peroxide for 10 minutes. After that, the cells incubated with anti-Ki67 Cell growth inhibition assay (1:1,000) antibody for one hour followed by incubation in Cells were seeded at density 10 cells/well in 96-well the HiDef Detection™ Amplifier and HiDef Detection™ plates, then the cells were treated next day with series HRP Polymer Detector for 10 minutes in each. The cells doses below and above their IC concentrations of then incubated with DAB-chromogen system for 3-5 KPT-330, 0.25, 0.5, 1, and 2μm/L and then further minutes followed by counterstain in hematoxylin. Scoring incubated for 24, 48 and 72 hours respectively. Thereafter, of Ki67 was done depending on the percentage of positive the cells were subjected to cell proliferation analysis in cells (0% - 100%) of the total cells numbers in high-power consecutive days using MTT [3-(4,5-dimethylthiazol-2- fields, as in previous study (Zu et al., 2012). yl)-2,5-diphenyltetrazolium bromide] assay and incubated for 4 hours. After that, the cells were lysed by 200 µL Statistical analysis of DMSO. Then the spectrophotometric absorbance of In this study, all analysis was performed using the samples was determined using a microplate reader SPSS version 18 statistical software program (SPSS (Bio-Rad) (Ferreiro-Neira et al., 2016). Inc., Chicago, IL, USA). Chi-square and Fisher ’s exact tests were used to find the association between Colony formation assay the overexpression of XPO1, NF-κB and Ki67 with The ability of KPT-330 to inhibit the HT29 cell clinico-histopathological factures. Mann-Whitney U lines colonies formation was assessed by the colony test was used with continuous variables. All cell culture formation assay as described previously (Niu et al., experiments were performed in triplicate and repeated at 2015). Briefly, 10 HT29 cells were seeded into 24-well least three times while the one-way ANOVA was used to plates in triplicates and then treated with vehicle control compare the mean between groups. The P values <0.05 (DMSO) and series doses of KPT-330, 0.5, 1, 2µmol/L) was considered statistically significant difference. for 24 hours. The culture medium was changed and the cells were incubated to allow colonies formation. After Results two weeks, cells were fixed with 5% glutaraldehyde in PBS and stained with 0.1% crystal violet. The numbers Expressions of XPO1, NF-κB and Ki67 in CRC of colonies formed were confirmed by manual counting The average age of the patients was 49.9 years (range for each dose. 24-80), with 55% (22/40) were females. After we used the IHC analysis, the majority of XPO1 expressions were Immunocytochemical (ICC) expressions of Ki67 in HT29- nuclear and/or nuclear membrane with weak to moderate KPT-330-treated cells diffuse cytoplasmic staining. The XPO1 expression was The HT29 cells seeded on cover slips in 6-well identified and scored as negative (15%), weak (12.5%), at density of 10 cells, incubated overnight and then moderate (20%) and strong (52.5%). The only strong score subjected to increasing doses of KPT-330 and incubated was considered as XPO1 overexpression (52.5%) while Figure 1. XPO1 Expression in Adjacent Normal Epithelium (A1 and B) and Tumor Cells (A2 and C). (Original Magnification- 200x and 400x, respectively). Asian Pacific Journal of Cancer Prevention, Vol 20 3749 Mohammed Aladhraei et al Figure 2. Association of XPO1 Overexpression and NF-κB with Ki67. The IHC expressions of both XPO1 (A) and NF-κB (B) show highly concordance of immunoreactivity within the tissue sections expressed Ki67 (C and D respectively) (original magnification- 400x). the 47.5% were considered as XPO1 non-overexpression. Although there was no big differences in the frequency of the XPO1 immunostaining between the tumor cells and normal adjacent epithelium, the intensity of XPO1 expression was abundant, intense and more apparent in tumor cells compared to the adjacent normal epithelial with significant difference (P<0.001) (Table 1 and Figure 1). The positive NF-κB expression was noted in 32.5% (13/40) while the Ki67 was observed as high expression in 72.5% (29/40) of CRC tumors (Table 2). Association between the XPO1 overexpression and NF-κB, Ki67 with clinicopathological features of CRC tissue samples In this study we identified a high tendency of XPO1 overexpression in moderately/poorly differentiated tumors and was significantly associated with advanced tumor stage (III/IV) (P=0.059 and P=0.049, respectively) (Table 2). Although the XPO1 overexpression was frequently noted in tumors with increasing numbers of positive lymph nodes metastasis, this tendency did not reach to the statistical significance difference (P=0.308). Significantly, we found a strong concordance between the XPO1 overexpression and high Ki67 expression in the tumor cells within the most tissue sections that showed immunoreactivity for both XPO1 and Ki67, and the XPO1 Table 1. Expression of XPO1 in CRC Tumors Cells and Adjacent Normal Epithelium XPO1 Number of patients (%) P value Figure 3. Correlation between the XPO1 overexpression expression Normal (%) Cancer (%) and Ki67 with tumor size. Mann-Whitney test demonstrate Negative 3 (7.5) 6 (15.0) that the tumors with larger size exhibit significant high Ki67 expression (A) (P=0.012) and to some extent the Weak 18 (45.0) 5 (12.5) XPO1 overexpression (B) (P=0.168). * represents the Moderate 15 (37.5) 8 (20.0) significant statistical differences between groups at P<0.05. Strong 4 (10.0) 21 (52.5) <0.001 3750 Asian Pacific Journal of Cancer Prevention, Vol 20 DOI:10.31557/APJCP.2019.20.12.3747 XPO1 Overexpression with NF-κB and Ki67 in CRC Figure 4. KPT-330 Inhibits the Growth and Proliferation of HT29 Cell Line. MTT assay (A), note the decreases of rate cell growth after 24, 48 and 72 hours of incubation while the colony formation assay (B) evince the ability of KPT-300 to inhibit the colonies formation. The ICC (C) staining shows reversible results of cell-expressed-ki67 and KPT-330 at doses 0.5 to 2µmol/L. *,** represent the statistical differences between the treated and untreated cells (P<0.001 and P<0.0001, respectively). overexpression was strongly associated with the high Ki67 the high Ki67 expression (P=0.007) (Table 2 and Figure 2) expression (P=0.001) (Table 2 and Figure 2). On the same manner, the CRC tumors sizes in this study ranges from KPT-330 inhibits cell proliferation, colony formation and 3 to 11 cm with mean 6.6 cm and we noted that the high Ki67 expression of HT29 cells nuclear Ki67 expression was associated with an increase As shown at Figure 4A, the increased of KPT-330 serial tumor size (P=0.012) but not XPO1 (P=0.168), with concentrations from 0.25, 0.5 and 1 to 2 µmol/L induced Mann-Whitney test, as it is illustrated in Figure 3. In this the cells growth inhibition after 72 hours of incubation. study, although the XPO1 overexpression was noticed in The IC value was 0.9 µmol/L in HT29 cells. These results 8/13 of positive NF-κB expression tumors, the statistical revealed that, when the KPT-330 dose exceeds 1 µmol/L significant association between the XPO1 overexpression and the action of time was >24 hours, the growth of cells and NF-κB was not found (P=0.427) (Table 3). However, was inhibited in a time and dose-dependent manner. most of the NF-κB positive tumors (11/13) were involved Further evaluation of the long-term effect of KPT-330 with positive lymph nodes metastasis with significant on HT29 growth, the clonogenic assay was performed difference (P=0.027) and showed strong association with for 2 weeks. The results revealed that the colonies Table 2. Association of XPO1 Overexpression and NF-κB with Clinicopathological Features and Ki67 Expression Clinicopathological Patients XPO1 expression P-value NF-κB expression P-value features and Ki67 expression No (%) Nonoverex-pressio Overexpr-ession -ve +ve Tumor differentiation 0.059 0.906 Well 19 (47.5) 12 7 13 6 Moderate/Poor 21 (52.5) 7 14 14 7 Lymph node metastasis 0.366 0.027* Negative 16 (40) 9 7 14 2 Positive 24 (60) 10 14 13 11 Tumor stage 0.049* 0.286 I–II 11 (27.5) 8 3 4 7 III-IV 29 (72.5) 11 18 7 22 Ki67 0.001** 0.007** <40% 11 (27.5) 10 1 11 0 ≥40% 29 (72.5) 9 20 16 13 *,** represent the statistical P<0.001 and P<0.0001, respectively. Asian Pacific Journal of Cancer Prevention, Vol 20 3751 Mohammed Aladhraei et al CRC tissues which may attribute to the large differences in Table 3. Association between the XPO1 Overexpression and NF-κB in CRC the sample sizes in each of well and poorly differentiated tumor groups; others were found such association in other NF-κB Patients XPO1 expression P value types of cancers such as ovarian cancer (Noske et al., expression No (%) Nonoverex- Overexpr- 2008), glioma (Shen et al., 2009) and the XPO1 expression pressio ession was increased from well to poorly differentiated breast Negative 27 (67.5) 14 13 tumors (Yue et al., 2018). Additionally, we noted that the Positive 13 (32.5) 5 8 0.427 poorly differentiated tumors were significantly increased numbers of metastatic lymph nodes, which goes in line formation decreased significantly by 86%, 52% and 21% with a cohort study results of 124,180 CRC patients by in response to KPT-330 doses of 0.5, 1 and 2 μmol/L, Ricciardi et al., (2006) who concluded that the poorly respectively (Figure 4B). Moreover, the Ki67 ICC differentiated tumors were much more likely to be with staining of KPT-330-treated-HT29 cells showed that the lymph node positive than well-differentiated tumors. Ki67 positive cells in the KPT-330 treated groups was In this study, most of moderately/ poorly differentiated significantly reduce compared to the untreated (Figure tumors with XPO1 overexpression were involved by 4C). These results were consistent with the MTT and lymph node metastasis. Moreover, the clinical trial colony formation assays results, confirming that the carried by Mau-Soerensen et al., (2014) among advanced KPT-330 inhibits the proliferation of HT29 colon cancer metastatic CRC patients and used oral KPT-330 for 28 cell line in dose and time dependent manner. days confirmed the valuable of XPO1 inhibitors in line of disease stability for advanced staged CRC patients. Collectively, these results may add insights of XPO1 Discussion involvement in the CRC pathogenesis and progression XPO1 is the major mammalian exporter protein that leading to poorly differentiated tumors and advance tumor facilitates the nucleocytoplasmic transport of over 200 stages. In some tumors such as lung cancer and mantle cell tumor suppressors and cell cycle regulatory proteins. Its lymphoma cells, the XPO1 inhibitors modulate the NF- overexpression reported in several types of tumors and was κB activity through trapping of IκB in the nucleus, which correlated with aggressive behavior and poor survival (Sun is target of XPO1 for nucleocytoplasmic transportation, et al., 2016). Furthermore, in recent years the XPO1 gains a leading to repression of NF-κB activity over time which great attention after launching a next-generation selinexor contributes in the growth suppression and apoptosis (KPT-330) as selective inhibition of nuclear export of induction (Zhang et al., 2013). In this study we found that XPO1 with good properties regarding low toxicity in the XPO1 overexpression was associated, to some extent, vivo and good tolerability by the patients (Lapalombella with the NF-κB positive expression without significant et al., 2012; Hing et al., 2016). In CRC, little is known difference. On the other hand, it has been proved that about the overexpression of XPO1 in tissue samples and the activated NF-κB contributes in the progression of its association with histopathological features, NF-κB CRC through upregulation expression of diverse target and Ki67. Our previous study concerned of p53, reported genes that are involved in inflammation (cytokines), cell that the XPO1 positivity was associated with loss of p53 proliferation (Cyclin D1), angiogenesis (VEGF, IL-8, expression in CRC tumors with lymph node metastasis COX2), and metastasis (MMP9) (Wang et al., 2009; Xie (Aladhraei et al., 2019). In the current study, we noticed et al., 2019) make it an interesting tumor marker in CRC a significant apparent of XPO1 overexpression in CRC pathogenesis. In this regard, the results of our study showed tumor cells compared to the adjacent normal epithelium that the NF-κB expression was significantly associated as it was reported in many other types of cancers such as with positive lymph node metastasis. Furthermore, strong esophageal (van der Watt et al., 2014), gastric (Subhash et significant association between the expressions of NF-κB al., 2018), lung (Gao et al., 2015), and ovarian (Noske et and high Ki67 expression was identified. These findings al., 2008) cancers, as well as leukemic cells (Kojima et al., were consistent with in vitro studies (Lu et al., 2016) as 2013). The overexpression of XPO1 within the tumor cells well as with NF-κB activity role in CRC tumors (Meteoglu may reflect its abundance and suggests a gain-of-function et al., 2015). These results add an insight in line of NF-κB or oncogenic activity in line of further CRC pathogenesis involvement in CRC metastasis and proliferation makes a (Conforti et al., 2015). Crochiere et al., (2016) suggested rational for targeting of NF-κB in CRC as it was reported that the abundance of XPO1 within the tumor cells might (Sakamoto and Maeda, 2010). It is well known that the be able to predict the drug resistance. sustained proliferation is one of the cancer hallmarks As it is known, the tumor grade is considered as acquired during cancer development and progression stage-independent prognostic factor in CRC, and the (Hanahan and Weinberg, 2011). In this regard, we poorly differentiated tumors are associated with poor identified that the overexpression of XPO1 was strongly patient survival (Fleming, et al., 2012). In this study associated with Ki67 expression, which in turn reflected we identified a high tendency of XPO1 overexpression the implication of XPO1 overexpression in mislocalization towards the moderate/poorly differentiated tumors as well of essential cell cycle inhibitory proteins such as p27, as increase of XPO1 overexpression frequency in advance p53, cyclins and some apoptotic proteins (Nguyen et al., tumor stages III-IV with significant difference. Although 2012; Niu et al., 2015) lead to unregulated cell division Shintani et al., (2016) studied the XPO1 expression in and increased tumor size. Despite ascending tendency CRC tissue samples, they did not find such association in of XPO1 expression was noticed with increased tumor 3752 Asian Pacific Journal of Cancer Prevention, Vol 20 DOI:10.31557/APJCP.2019.20.12.3747 XPO1 Overexpression with NF-κB and Ki67 in CRC size, our study did not find a direct association between XPO1 overexpression prognostication in CRC patients the XPO1 overexpression and tumor size; while the high may be recommended. Ki67 expression was significantly noticed in larger tumor sizes. The strong association between the overexpression Acknowledgements of XPO1 and high Ki67 expression in CRC patients’ tumors was in concordance with our in vitro results that The authors are grateful to the Department of confirmed the anti-proliferative effect of KPT-330. The Pathobiology, Faculty of Science, Mahidol University KPT-330 induced the growth inhibition and suppressed for the providing the laboratory space and facilities as the HT29 colorectal cancer cell line proliferation in dose well as the excellent technical assistance assisted by and time dependent manner as shown by the MTT assay. Mrs. Pranom Puchadapirom and Mrs. Maliwan Emyeam. Furthermore, the experiments showed a great stability of Ethically, the study protocol followed the guidelines KPT-330 in vitro, which means a continuous inhibition approved by the Ethical Committee of the Medical of growth over the time during incubation and persisted Research of the National Health and Medical Research for up to 72 hours. This explains the powerful binding of Committee (NHMRC), Republic of Yemen (Approval No. KPT-330 in slowly reversible action with XPO1 leading B2/10-2017). No potential conflict of interest relevant to to inhibition the binding between the cargoes proteins this article was reported. and XPO1 in HT29 cancer cell line which in turn induce nuclear retention of cell cycle regulatory proteins, inhibits References the proliferation and may initiate the apoptosis (Draetta et Abdul Razak AR, Mau-Soerensen M, Gabrail NY, et al (2016). al., 2011; Senapedis et al., 2014). The long-term effect of First-in-class, first-in-human phase i study of selinexor, KPT-330 on the HT29 colony formation was significantly a selective inhibitor of nuclear export, in patients with seen as a decrease of colonies formation with increase advanced solid tumors. J Clin Oncol, 34, 4142-50. of KPT-330 concentration. These findings were further Aladhraei M, Al-Salami E, Poungvarin N, Suwannalert P (2019). supported by the evaluation of Ki67 expression by ICC in The roles of p53 and XPO1 on colorectal cancer progression HT29-KPT-330-treated cells. 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Published: Dec 1, 2019

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