UNC3866

Multiple myeloma driving factor WHSC1 is a transcription target of oncogene HMGA2 that facilitates colon cancer proliferation and metastasis

Hou-Hsien Liu a, Chia-Hwa Lee b, Yi-Chen Hsieh c, d, e, Duen-Wei Hsu f, Er-Chieh Cho a, g, h, *

A B S T R A C T

Colon cancer is a common human cancer worldwide. The survival rate of late staged or metastatic colon cancer patients remains low even though the effectiveness of treatment in colon cancer has greatly improved. Research on tumorigenesis mechanisms and discovery of novel molecular target for treating colon cancer is critical.
The promotion roles of WHSC1 in multiple myeloma have been demonstrated previously, yet, the regulation of WHSC1 in other cancers is largely unknown, especially in colon cancer. Here, in this study, we analyzed and identified WHSC1 while studying the genetic regulations of HMGA2 in colon cancer cells by microarray analysis, and investigated the HMGA2-WHSC1 interaction. We then applied CRISPR technology to establish stable WHSC1 knockout cells, to address the functional regulation of WHSC1 in colon cancer. In summary, our results for the first time identified the HMGA2-WHSC1 interaction in colon cancer. Moreover, we discovered that WHSC1 promotes cancer proliferation, facilitates resistance of chemotherapy agent, and promotes metastatic capacity of colon cancer.

Keywords: WHSC1 HMGA2
Colon cancer Metastasis
Transcription regulation

1. Introduction

Colon cancer is one of the most common cancers in human, especially in western countries. Common risk factors of colon cancer include unhealthy life style such as being overweight, physically inactive, smoking, high red meat diet, and heavy alcohol use, etc [1e4]. Chromosomal instability is also a critical pathway which promotes tumorigenesis of colon cancer [5e7]. Despite the improvement of therapeutic development in the past decades, there are still challenges for treating late staged and metastatic patients, and therefore, it is critical to decipher and investigate the molecular regulatory mechanisms controlling tumorigenesis in colon cancer. Wolf-Hirschhorn Syndrome Candidate 1 (WHSC1), also called multiple myeloma (MM) SET domain, MMSET, and histone-lysine N-methyltransferase, NSD2, is a key factor associated with Wolf- Hirschhorn syndrome [8,9]. WHSC1 belongs to NSD family, which harbors a SET domain and exhibits histone methyltransferase ac- tivity [10,11]. It is well-studied that around 15e20% of MM patients with poor disease prognosis is mediated by elevated of expression of WHSC1 protein which occurs in all the MM patients with t(4; 14) translocation [8,9,12,13]. Moreover, WHSC1 was suggested to facilitate drug resistance in cancer cells through promoting DNA repair mechanisms, and inhibit apoptosis pathway through regu- lating B cell lymphoma-2 (Bcl-2) anti-apoptotic protein in cancer cells [14,15]. Moreover, a recent study demonstrated that WHSC1 plays a role in immune infiltration regulation in prostate cancer [16]. Overexpressed WHSC1 was found associated with poor prognosis and/or participated in pathogenesis of various cancers, include neuroblastoma, colon cancer, hepatocellular carcinoma, prostate cancer and head and neck cancer [17,18]. However, the transcriptional regulation of WHSC1 in cancer is largely unknown, especially, no one has investigated it in colon cancer.
High-mobility group AT-hook 2 (HMGA2) is a member of the high mobility group family with transcription factor capacity which normally expresses during early development of embryos only but not in adults [19,20]. Inactivation of HMGA2 resulted in pygmy phenotype in mice, and a HMGA2 common variant was identified associated with height in human [21,22]. Overexpression of HMGA2 was found in various progressive neoplasms, where HMGA2 pro- motes proliferation and malignant transformation of cancers as an oncogene [20,23,24]. In this study, WHSC1 was identified in the microarray experiment when analyzing HMGA2 overexpressed DLD-1 colon cancer cells. The association between HMGA2 and WHSC1 was studied, and the regulation of WHSC1 in colon cancer tumorigenesis was also investigated.

2. Materials and methods

2.1. Cell culture and stable cells establishment

HCT116 and DLD-1 colon cancer cells (ATCC, USA) were grown in RPMI with 10% Fetal Bovine Serum and 1% Antibiotic-antimycotic (Gibco, USA) in 37 ◦C, 5% CO2 incubator. Cells were infected with lent-viruses containing clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas9) plasmids that were utilized to knockout WHSC1, named C-WHSC1, or scramble control, named C-scramble, and then puromycin (Cayman Chemical, USA) was used for stable cells selection.

2.2. Plasmids, siRNAs, and transfection

Plasmids used for overexpression of HMGA2 (HMGA2-GFP) and WHSC1 (WHSC1-DDDDK) were from OriGene (Rockville, USA). Short interference RNAs (siRNAs) were from Santa Cruz Biotech- nology (Dallas, USA). Transfection reagent (T-Pro, Taiwan) was applied for transfection of DNA plasmids or siRNAs. Cells were then harvested 48e72 h later for assessment [25].

2.3. Polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR)

For PCR, 2X SuperRed PCR Mix was applied (Tools, Taiwan). PCR reaction was carried out as suggested by the manufacturer’s in- structions for SENSQUEST labcycler, and then samples were analyzed by DNA electrophoresis. For qPCR, 2X SYBR qPCR Mix was applied (Tools). Reaction was performed as suggested by the manufacturer’s instructions for StepOnePlus Real-Time PCR System [26]. Primers applied in qPCR can be provided upon request.

2.4. CRISPR/Cas9 plasmid construction and lentiviral production

Lentiviral particles were produced by transient transfection of Phoenix-ECO cells (CRL-3214) usingTransIT®-LT1 Reagent (Mirus Bio LLC, USA). Guide oligonucleotides were phosphorylated, annealed, and cloned into the BsmBI site of the lentiCRISPR v2 vector (Addgene, kindly provided by Feng Zhang) according to the Zhang laboratory protocol (F. Zhang lab, MIT, USA). All the plasmid constructs were verified by sequencing.ThelentiCRISPRconstruct was co-transfected with pMD2.G (Addgene, USA) and psPAX2 (Addgene, both kindly provided by Didier Trono, EPFL, Lausanne, Switzerland). Lentiviral particles were collected at 36 and 72 h and then concentrated with a Lenti-X Concentrator® (Clontech, USA).

2.5. Sanger sequencing and gene editing efficiency assay

Genomic DNA was extracted, and the WHSC1exon 4region was PCR-amplified using the following primers: forward CCTTTCTGTTCAGAGTGTTGTAA and reverse CTCCAGCAAGGAGTCATATT. The PCR product of 514 base-pair was purified using a PCR Clean-up Purification Kit and sequenced by Sanger sequencing using the forward PCR primers. The editing efficiency of the sgRNAs and the potential induced mutations were assessed using TIDE software (https://tide-calculator.nki.nl; Netherlands Cancer Insti- tute), which required only two Sanger sequencing runs from wild- type cells and mutated cells.

2.6. Western blot assay

For protein expression analysis, cells after the treatment were harvested. Proteins were extracted and separated by SDS-PAGE. Polyvinylidene difluoride (Pall, USA) membrane was applied. GAPDH, HMGA2, and vinculin antibodies were from GeneTex (CA, USA); WHSC1 antibody was from Santa Cruz. Finally, enhanced chemiluminescene (ECL, Advansta) was applied to visualize the signals [27].

2.7. Luciferase reporter assay

Reporter assay was carried out as described previously [28]. WHSC1 promoter construct contains about 1 kb sequence of WHSC1 promoter (S702150, Lightswitch, ActiveMotif, USA). 48 h later after the transfection, cells were harvested and luciferase ac- tivity was measured according to the manufacturer’s instructions (Lightswitch). Each treatment was done as triplicate, and n ¼ 3 for the assay.

2.8. Electrophoretic mobility shift assay (EMSA)

EMSA was performed with recombinant HMGA2 proteins (ProSpec, Israel) and WHSC1 promoter region (S702150, Light- switch, ActiveMotif, USA). WHSC1 promoter was obtained by PCR using forward primer GAATAGTCTTCCTGCCTCCCC and reverse primer CCAGTGGTTCTAGGACAGGC, resulting in a 924bp PCR product. The binding buffer include 25 mM HEPES, 0.5 mM EDTA (pH8), 50 mM KCl, 10% glycerol, 50 nM DTT, and 50 nM PMSF. PCR product was incubated with recombinant proteins at RT for 30 min, and then samples were separated by 1% agarose gel. Gel was then applied in EtBr DNA staining, and UV was applied for DNA detection.

2.9. MTS cell proliferation assay

Cells were seeded in 96 well plates, and cell proliferation was analyzed at indicated time point after the treatments according to the manufacturer’s instructions (Promega, USA) in MTS assay [26]. Each treatment was done as triplicate, and n ¼ 3 for the assay.

2.10. Wound healing assay

Cells were seeded in the 6 or 12 well plates, and the assay was performed as described [29]. Migration rate is definite as (0 h dis- tance e each time point distances)/0 h distance. The assay was performed as triplicate for each condition.

2.11. Colony formation assay

100 cells per well were seeded in 6 well culture plate, and after the treatment, cells were then grew in the incubator for 10e12 days and analyzed as described [30]. The assay was performed as trip- licate for each condition.

2.12. Transwell cell invasion assay

The assay was performed according to the manufacturer’s in- structions (Millipore) and as described [25]. Cells were mixed with serum free medium and seeded in the upper layer of the transwell insert, while lower layer contained 10% FBS in medium. Matrigel was applied in the upper layer. At the end of the experiment, membranes were fixed and then stained with 0.5% crystal violet for cell visualization.

2.13. Statistical analysis

The comparison between the measurements obtained for each group was performed by Student’s t-test. P-value<0.05 was considered to reach a significant difference. 3. Results 3.1. HMGA2 & WHSC1 expression correlated in colon cancer cells While studying the role of HMGA2 in genetic regulation in colon cancer, we overexpressed control or HMGA2 plasmids in colon cancer cells DLD-1, sent the extracted RNA samples for microarray analysis, and set the 1.5 fold threshold selection condition in microarray data. Among the genes altered along with HMGA2 overexpression, WHSC1, an oncogenic gene in MM, was identified and caught our eyes. The expression level of WHSC1 within control and HMGA2 overexpressed samples analyzed through microarray was quantified (Fig. 1a). Next, to further verify the WHSC1 gene alteration mediated by HMGA2, cells were transfected with HMGA2 (or control) plasmids, and then qPCR assay was performed to examine the expressions of HMGA2 and WHSC1 at the gene level (Fig. 1b). Results showed that when there was upregulated HMGA2, there was elevated level of WHSC1 in colon cancer cells. Next, we further investigated the expression correlation be- tween HMGA2 and WHSC1 at the protein level. HMGA2 over- expression plasmids and siRNA were applied in DLD-1 cells for transfection, and then the protein levels of WHSC1 were analyzed by Western blot assay (Fig. 1c and d). Our results indicated that WHSC1 proteins were upregulated along with the HMGA2 over- expression (Fig. 1c), while as WHSC1 levels were reduced when HMGA2 was knockdown in cells (Fig. 1d). The data demonstrated the expression association between HMGA2 and WHSC1 at protein level in colon cancer cells. 3.2. WHSC1 is a putative transcription target of HMGA2 in colon cancer cells HMGA2 harbors transcription factor capacity, and therefore we further examined whether WHSC1 could be a downstream tran- scription target of HMGA2. Luciferase reporter assay was carried out to examine the regulation of WHSC1 promoter by HMGA2 in colon cancer DLD-1 and HCT116 cells. Either control or HMGA2 plasmids were co-transfected into colon cancer cells with WHSC1 promoter construct, and the activity of the luciferase was measured as described in the materials and methods section. The results showed that HMGA2 overexpression led to elevated luciferase ac- tivity of WHSC1 promoter compared to control in both HCT116 (increased about 3 folds) and DLD-1 (increased about 1.5 folds) cells (Fig. 2a and b), suggested that HMGA2 can activate WHSC1 at the transcription level. Next, we investigated the possibility of a physical interaction between HMGA2 protein and WHSC1 promoter region by electrophoretic mobility shift assay (EMSA), as described in the materials and methods. Our results showed that PCR product containing WHSC1 promoter region bond to recombinant HMGA2 proteins and therby formed complex (Fig. 2c). The data suggested a physical interaction between HMGA2 proteins and WHSC1 pro- moter for facilitating the transcription activation of WHSC1 pro- moter, and demonstrates that WHSC1 as a putative downstream target of the oncogene HMGA2. 3.3. Development and characterization of CRISPR/Cas9-mediated WHSC1 knockout cells In order to investigate the functional regulation of WHSC1 in colon cancer, we utilized the CRISPR/Cas9 technology for WHSC1 gene knockout in DLD-1 cells. The utility of CRISPR/Cas9 genome editing in DLD-1 cells was investigated by targeting custom- designed protospacer on the WHSC1 locus. As shown in the genomic map (Fig. 3a), protospacer targets the negative strand of chromosome 4p.11 of WHSC1 gene locus. Control scrambled lenti- virus was applied for transduction of cells and the production of a wild-type WHSC1 sequence and showed no evidence of gene editing (Fig. 3b). At the same time, the WHSC1sgRNA lentivirus was applied for transduction of cells and the production of multiple gene disruptions (red arrowhead sites) (Fig. 3c). In addition, WHSC1sgRNA delivery analyzed by TIDE software showed signifi- cantly 74% gene editing efficiency compared to control (Fig. 3d). Scramble control cells (C-scramble) and WHSC1-edited cells (C-WHSC1) were established, and the protein expression level of WHSC1 in these cell lines were examined by Western blot assay (Fig. 3e), demonstrated that WHSC1 expression was depleted in C- WHSC1 cells. This pair of CRISPR cells was then applied in the following experiments. 3.4. WHSC1 promotes cancer cells proliferation and resistance of chemotherapy Firstly, we characterized the proliferation rate of CRISPR cells, to examine the impacts of WHSC1 expression towards cancer cell proliferation by MTS assays. We also studied the role of WHSC1 in colon cancer proliferation under treatments of chemotherapy agent 5-fluorouracil (5FU). The results showed that C-WHSC1 cells grew slower compared to C-scramble cells, implying that depletion of WHSC1 reduced cell proliferation, and 5FU treatment further decreased the proliferation of cells (Fig. 4a), suggesting that depletion of WHSC1 could sensitize 5FU treatment in colon cancer inhibition. Further, colony formation assay was carried out to examine the long-term cell proliferation capacity. CRISPR cells were applied in the assay, and consistent with data from MTS assays, results from colony assay showed that WHSC1 knockout led to reduced colony numbers, and 5FU treatment further decreased the capacity of C- WHSC1 cells in forming colonies (Fig. 4b). The numbers of colonies from C-scramble and C-WHSC1 were analyzed. Control or WHSC1 overexpression experiments were also performed in colony for- mation assays, and there was a compatible trend, though, not sta- tistically significant, of the impacts of WHSC1 in increasing colony numbers (data not shown). The above data suggested that WHSC1 promotes cancer cells proliferation and resistance of chemotherapy. 3.5. WHSC1 facilitates colon cancer metastasis Migration and invasion capacities of the cancer cells are critical for tumor metastasis. WHSC1 was found involved in migration in other cancers previously, and therefore it was of our great interest to investigate the role of WHSC1 in colon cancer migration. CRISPR cells were applied in the wound healing assay, and the results showed that C-WHSC1 cells migrated slower than C-scramble cells (Fig. 4c). It indicates that downregulation of WHSC1 could suppress colon cancer migration. Moreover, WHSC1 expressing plasmids were transfected in this assay, and compatibly, the results indicated that cells with WHSC1 overexpression migrated faster than control cells (data not shown). Migration and invasion properties of the cancer cells are very often connected and can both facilitate tumor metastasis, therefore, we next applied CRISPR cells in the invasion assay. The results showed that depletion of WHSC1 reduced the number of invaded cells compared to control C-scramble cells (Fig. 4d). The above re- sults suggested that WHSC1 promotes metastasis capacity of colon cancer. 4. Discussion Despite the fact that WHSC1 being a driving factor in MM tumor pathogenesis as previously demonstrated, the functional roles of WHSC1 and the upstream regulation of WHSC1 in cancer remain largely unknown. As mentioned, HMGA2 was identified as an oncogene with transcription factor properties. Here in this study, we report the first time evidence of oncogene HMGA2 as a novel upstream regulator of WHSC1 (Figs. 1e2). HMGA2 has been sug- gested as an oncogene in various cancers, yet largely is still un- known about the regulatory mechanisms of HMGA2 in cancer progression. Identifying WHSC1 as a putative novel downstream target of HMGA2 fills in the gaps in knowledge of the pivotal role of HMGA2 in oncogenesis, and further investigation between HMGA2 and WHSC1 is awaited. Challenges are existing for colon cancer treatment in clinical sides. For example, KRAS mutated colon cancer represents 30%e 50% of total colon cancer patients, and KRAS mutation leads to no response towards anti-epidermal growth factor receptor (EGFR)- targeted therapy [31e34]. Here in this study, both HCT116 and DLD- 1 cells we applied are KRAS mutant colon cancer cell lines. Even though further study is required, our results provide another possible breakthrough point for future KRAS mutant colon cancer treatment using WHSC1 inhibitors as a therapy option. Increased expression of WHSC1 has been found in various cancer types, however, except for MM and prostate cancer, little of the molecular mechanisms of WHSC1 in tumorigenesis was investigated. To our knowledge, we are the first to apply CRISPR technology in studying the role of WHSC1 in colon cancer pro- gression (Fig. 3). With the CRISPR cell lines, we characterized and demonstrated the positive impacts of WHSC1 in cancer prolifera- tion, resistance in chemotherapy, and first time in metastasis (Fig. 4). In conclusion, this study suggested the role of HMGA2-WHSC1 interaction in colon cancer tumorigenesis, and provided the novel insights of the regulations of HMGA2 in tumorigenesis. This study also demonstrated the functional impacts of WHSC1 in colon can- cer pathogenesis, and suggested that depletion of WHSC1 along or as adjuvant with standard chemotherapy agents could benefit future clinical treatments of colon cancer. WHSC1 is suggested as a target for colon cancer therapy. References [1] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int. J. Canc. 136 (5) (2015) E359eE386. [2] C. Jochem, M. Leitzmann, Obesity and Colorectal Cancer. Recent results in cancer research Fortschritte der Krebsforschung Progres dans les recherches sur, le cancer 208 (2016) 17e41. [3] A. Richardson, J. Hayes, C. Frampton, J. Potter, Modifiable lifestyle factors that could reduce the incidence of colorectal cancer in New Zealand, N. Z. Med. J. 129 (1447) (2016) 13e20. [4] S.E. Steck, M. Guinter, J. Zheng, C.A. Thomson, Index-based dietary patterns and colorectal cancer risk: a systematic review, Advances in nutrition 6 (6) (2015) 763e773. [5] J. Bogaert, H. Prenen, Molecular genetics of colorectal cancer, Ann. Gastro- enterol. 27 (1) (2014) 9e14. [6] J.C. Obuch, D.J. Ahnen, Colorectal cancer: genetics is changing everything, Gastroenterol. Clin. N. Am. 45 (3) (2016) 459e476. [7] M. Wright, J.S. Beaty, C.A. Ternent, Molecular markers for colorectal cancer, Surg. Clin. 97 (3) (2017) 683e701. [8] I. Stec, T.J. Wright, G.J. van Ommen, P.A. de Boer, A. van Haeringen, A.F. Moorman, M.R. Altherr, J.T. den Dunnen, WHSC1, a 90 kb SET domain- containing gene, expressed in early development and homologous to a Drosophila dysmorphy gene maps in the Wolf-Hirschhorn syndrome critical region and is fused to IgH in t(4;14) multiple myeloma, Hum. Mol. Genet. 7 (7) (1998) 1071e1082. [9] M. Santra, F. Zhan, E. Tian, B. Barlogie, J. Shaughnessy Jr., A subset of multiple myeloma harboring the t(4;14)(p16;q32) translocation lacks FGFR3 expres- sion but maintains an IGH/MMSET fusion transcript, Blood 101 (6) (2003) 2374e2376. [10] T. Vougiouklakis, R. Hamamoto, Y. Nakamura, V. Saloura, The NSD family of protein methyltransferases in human cancer, Epigenomics 7 (5) (2015) 863e874. [11] M. Morishita, E. di Luccio, Cancers and the NSD family of histone lysine methyltransferases, Biochim. Biophys. Acta 1816 (2) (2011) 158e163. [12] M.E. Issa, F.S. Takhsha, C.S. Chirumamilla, C. Perez-Novo, W. Vanden Berghe, M. Cuendet, Epigenetic strategies to reverse drug resistance in heterogeneous multiple myeloma, Clin. Epigenet. 9 (2017) 17. [13] J.J. Keats, C.A. Maxwell, B.J. Taylor, M.J. Hendzel, M. Chesi, P.L. Bergsagel, L.M. Larratt, M.J. Mant, T. Reiman, A.R. Belch, et al., Overexpression of tran- scripts originating from the MMSET locus characterizes all t(4;14)(p16;q32)- positive multiple myeloma patients, Blood 105 (10) (2005) 4060e4069. [14] M.Y. Shah, E. Martinez-Garcia, J.M. Phillip, A.B. Chambliss, R. Popovic, T. Ezponda, E.C. Small, C. Will, M.P. Phillip, P. Neri, et al., MMSET/WHSC1 enhances DNA damage repair leading to an increase in resistance to chemo- therapeutic agents, Oncogene 35 (45) (2016) 5905e5915. [15] Y. Wang, L. Zhu, M. Guo, G. Sun, K. Zhou, W. Pang, D. Cao, X. Tang, X. Meng, Histone methyltransferase WHSC1 inhibits colorectal cancer cell apoptosis via targeting anti-apoptotic BCL2, Cell Death Dis. 7 (1) (2021) 19. [16] M.Y. Want, T. Tsuji, P.K. Singh, J.L. Thorne, J. Matsuzaki, E. Karasik, B. Gillard, E. Cortes Gomez, R.C. Koya, A. Lugade, et al., WHSC1/NSD2 regulates immune infiltration in prostate cancer, J Immunother Cancer 9 (2) (2021). [17] H.R. Hudlebusch, E. Santoni-Rugiu, R. Simon, E. Ralfkiaer, H.H. Rossing, J.V. Johansen, M. Jorgensen, G. Sauter, K. Helin, The histone methyltransferase and putative oncoprotein MMSET is overexpressed in a large variety of human tumors, Clin. Canc. Res. : an official journal of the American Association for Cancer Research 17 (9) (2011) 2919e2933. [18] A. Kassambara, B. Klein, J. Moreaux, MMSET is overexpressed in cancers: link with tumor aggressiveness, Biochemical and biophysical research communi- cations 379 (4) (2009) 840e845. [19] H.R. Ashar, R.A. Chouinard Jr., M. Dokur, K. Chada, In vivo UNC3866 modulation of HMGA2 expression, Biochim. Biophys. Acta 1799 (1e2) (2010) 55e61.
[20] A.R. Young, M. Narita, Oncogenic HMGA2: short or small? Gene Dev. 21 (9) (2007) 1005e1009.
[21] X. Zhou, K.F. Benson, H.R. Ashar, K. Chada, Mutation responsible for the mouse pygmy phenotype in the developmentally regulated factor HMGI-C, Nature 376 (6543) (1995) 771e774.
[22] M.N. Weedon, G. Lettre, R.M. Freathy, C.M. Lindgren, B.F. Voight, J.R. Perry, K.S. Elliott, R. Hackett, C. Guiducci, B. Shields, et al., A common variant of HMGA2 is associated with adult and childhood height in the general popu- lation, Nat. Genet. 39 (10) (2007) 1245e1250.
[23] D. D’Angelo, P. Mussnich, C. Arra, S. Battista, A. Fusco, Critical role of HMGA proteins in cancer cell chemoresistance, J. Mol. Med. 95 (4) (2017) 353e360.
[24] M. Fedele, A. Fusco, HMGA and cancer, Biochim. Biophys. Acta 1799 (1e2) (2010) 48e54.
[25] C.Y. Yu, W.C. Chang, J.H. Zheng, W.H. Hung, E.C. Cho, Transforming growth factor alpha promotes tumorigenesis and regulates epithelial-mesenchymal transition modulation in colon cancer, Biochemical and biophysical research communications 506 (4) (2018) 901e906.
[26] E.C. Cho, M.L. Kuo, X. Liu, L. Yang, Y.C. Hsieh, J. Wang, Y. Cheng, Y. Yen, Tumor suppressor FOXO3 regulates ribonucleotide reductase subunit RRM2B and impacts on survival of cancer patients, Oncotarget 5 (13) (2014) 4834e4844.
[27] K.C. Lee, P.Y. Lo, G.Y. Lee, J.H. Zheng, E.C. Cho, Carboxylated carbon nano- materials in cell cycle and apoptotic cell death regulation, J. Biotechnol. 296 (2019) 14e21.
[28] K.C. Lee, Y.L. Chen, C.C. Wang, J.H. Huang, E.C. Cho, Refluxed esterification of fullerene-conjugated P25 TiO2 promotes free radical scavenging capacity and facilitates antiaging potentials in human cells, ACS Appl. Mater. Interfaces 11 (1) (2019) 311e319.
[29] M.H. Lin, J.S. Wang, Y.C. Hsieh, J.H. Zheng, E.C. Cho, NO2 functionalized coumarin derivatives suppress cancer progression and facilitate apoptotic cell death in KRAS mutant colon cancer, Chem. Biol. Interact. 309 (2019) 108708.
[30] C.H. Chen, Y.C. Hsieh, P.M. Yang, Y.R. Liu, E.C. Cho, Dicoumarol suppresses HMGA2-mediated oncogenic capacities and inhibits cell proliferation by inducing apoptosis in colon cancer, Biochemical and biophysical research communications 524 (4) (2020) 1003e1009.
[31] A.V. Kudryavtseva, A.V. Lipatova, A.R. Zaretsky, A.A. Moskalev, M.S. Fedorova, A.S. Rasskazova, G.A. Shibukhova, A.V. Snezhkina, A.D. Kaprin, B.Y. Alekseev, et al., Important molecular genetic markers of colorectal cancer, Oncotarget 7 (33) (2016) 53959e53983.
[32] C. Tan, X. Du, KRAS mutation testing in metastatic colorectal cancer, World J. Gastroenterol. 18 (37) (2012) 5171e5180.
[33] C. Lo Nigro, V. Ricci, D. Vivenza, C. Granetto, T. Fabozzi, E. Miraglio, M.C. Merlano, Prognostic and predictive biomarkers in metastatic colorectal cancer anti-EGFR therapy, World J. Gastroenterol. 22 (30) (2016) 6944e6954.
[34] R. Dienstmann, R. Salazar, J. Tabernero, Overcoming Resistance to Anti-EGFR Therapy in Colorectal Cancer, American Society of Clinical Oncology educa- tional book American Society of Clinical Oncology Meeting, 2015, pp. e149e156.