大腸直腸癌是台灣最常見的癌症，也是世界上造成癌症死亡的第三大原因。 RAS信號通路在大腸直腸癌的發生和發展中相當重要。 RAS信號傳導透過兩個主要途徑分別是促分裂原活化蛋白激酶（MAPK）和磷酸肌醇3激酶。發芽的同源物2（SPRY2）屬於發芽家族，對RAS / MAPK途徑具有抑制作用。 SPRY2也可以激活PTEN，從而抑制癌細胞的生長。
我們的實驗證明，SPRY2可以抑制KRAS原生型Caco-2細胞的增殖和遷移，但可以刺激細胞侵襲。然而，SPRY2並不影響KRAS突變SW480細胞以及轉染KRAS突變質體的Caco-2細胞的增殖、遷移、和侵襲。在動物實驗中，敲落SPRY2基因可以促進Caco-2 細胞的腫瘤生長。將敲落SPRY2的Caco-2細胞注射到小鼠皮下，對照組相比，異種移植的生長更快且更大，但有比較少的肌肉浸潤的現象。而西方點墨的結果顯示，SPRY2抑制E-鈣粘蛋白，並促進N-鈣粘蛋白的表達，顯示激活了上皮-間質轉化（EMT）的能力。
在Caco-2細胞株的微陣列分析中，穩定敲落SPRY2會抑制JAK2和MMP25的表現。使用即時聚合酶鏈式反應和蛋白質轉漬研究，證實了這兩種可能的影響。臨床研究發現，SPRY2的過表達，似乎和KRAS原生型大腸直腸癌患者的總體生存不佳有關（p = 0.138）。
總之，SPRY2可能通過抑制Ras / Raf / MAPK / ERK途徑，抑制Caco-2細胞（野生型KRAS）的增殖和遷移。另一方面，SPRY2可以刺激Caco-2細胞侵襲, 也許是透過誘導JAK2或MMP25來達成。動物實驗中的腫瘤生長結果支持SPRY2抑制細胞增殖的結果，而臨床癌症檢體免疫染色則暗示SPRY2為促進野生型KRAS大腸直腸癌發展的角色。

Colorectal cancer (CRC) is the most common cancer in Taiwan and is the third cause of cancer death worldwide. RAS signaling pathway is critical in the pathogenesis and progression of CRC. Two of RAS signaling are mitogen-activated protein kinases (MAPK) and phosphoinositide-3 kinase. SPRY2 belongs to the sprouty family with inhibitory effect on RAS/MAPK pathway. SPRY2 could also activate PTEN, resulting in growth inhibition of cancer cells.
We provide evidence that SPRY2 suppresses the proliferation and migration, but stimulates the invasion of KRAS wild-type Caco-2 cells in vitro. However, SPRY2 did not affect the proliferation, migration, and invasion of KRAS mutant-SW480 cells and Caco-2 cells transfected with KRAS mut plasmid in vitro. Xenografts of stable SPRY2 knockdown Caco-2 cells were larger with less muscle invasion than control cells. Western blot revealed that SPRY2 downregulates the E-cadherin, while upregulates N-cadherin, suggesting activation of epithelial-mesenchymal transition.
Microarray profiling showed that knockdown of SPRY2 downregulates the expression of JAK2 and MMP25. Immunohistochemical study of clinical cohort demonstrated a trend toward high SPRY2 expression with a poor cancer-specific survival in wild-type KRAS CRC patients (p = 0.138).
In conclusion, SPRY2 inhibits the proliferation and migration of Caco-2 cells with wild-type KRAS, probably though inhibition of Ras/Raf/MAPK/ERK pathway. On the other hand, SPRY2 stimulates invasion of Caco-2 cells, possibly through induction of JAK2 or MMP25. The results of tumor growth in xenograft mouse model supported the growth inhibitory effects of SPRY2, while the IHC results using clinical cancer specimens suggests the tumor-enhancer role of SPRY2 in wild-type KRAS CRC.

1. Arnold, M. et al. Global patterns and trends in colorectal cancer incidence and mortality. Gut 66, 683-691 (2017).
2. Muller, M.F., Ibrahim, A.E. & Arends, M.J. Molecular pathological classification of colorectal cancer. Virchows Arch. 469, 125-134 (2016).
3. Markowitz, S.D. & Bertagnolli, M.M. Molecular origins of cancer: Molecular basis of colorectal cancer. N. Engl. J. Med. 361, 2449-2460 (2009).
4. Shia, J., Klimstra, D.S., Bagci, P., Basturk, O. & Adsay, N.V. TNM staging of colorectal carcinoma: issues and caveats. Semin. Diagn. Pathol. 29, 142-153 (2012).
5. Weiser, M.R. AJCC 8th Edition: Colorectal Cancer. Ann. Surg. Oncol. 25, 1454-1455 (2018).
6. Henson, D.E. et al. The anatomy of the TNM for colon cancer. J. Gastrointest. Oncol. 8, 12-19 (2017).
7. Ogino, S. & Goel, A. Molecular classification and correlates in colorectal cancer. J. Mol. Diagn. 10, 13-27 (2008).
8. Singh, M.P., Rai, S., Pandey, A., Singh, N.K. & Srivastava, S. Molecular subtypes of colorectal cancer: An emerging therapeutic opportunity for personalized medicine. Genes & Diseases (2019).
9. Duineveld, L.A. et al. Symptomatic and Asymptomatic Colon Cancer Recurrence: A Multicenter Cohort Study. Ann. Fam. Med. 14, 215-220 (2016).
10. Nozawa, H. et al. High-risk Stage II Colorectal Cancers Carry an Equivalent Risk of Peritoneal Recurrence to Stage III. In Vivo 32, 1235-1240 (2018).
11. Wang, Y. et al. Gene expression profiles and molecular markers to predict recurrence of Dukes' B colon cancer. J. Clin. Oncol. 22, 1564-1571 (2004).
12. Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y. & Krasnow, M.A. sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 92, 253-263 (1998).
13. Kramer, S., Okabe, M., Hacohen, N., Krasnow, M.A. & Hiromi, Y. Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila. Development 126, 2515-2525 (1999).
14. de Maximy, A.A. et al. Cloning and expression pattern of a mouse homologue of drosophila sprouty in the mouse embryo. Mech. Dev. 81, 213-216 (1999).
15. Leeksma, O.C. et al. Human sprouty 4, a new ras antagonist on 5q31, interacts with the dual specificity kinase TESK1. Eur. J. Biochem. 269, 2546-2556 (2002).
16. Gross, I., Bassit, B., Benezra, M. & Licht, J.D. Mammalian sprouty proteins inhibit cell growth and differentiation by preventing ras activation. J. Biol. Chem. 276, 46460-46468 (2001).
17. Impagnatiello, M.A. et al. Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells. J. Cell Biol. 152, 1087-1098 (2001).
18. Masoumi-Moghaddam, S., Amini, A. & Morris, D.L. The developing story of Sprouty and cancer. Cancer Metastasis Rev. 33, 695-720 (2014).
19. Minowada, G. et al. Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed. Development 126, 4465-4475 (1999).
20. Ding, W., Bellusci, S., Shi, W. & Warburton, D. Genomic structure and promoter characterization of the human Sprouty4 gene, a novel regulator of lung morphogenesis. Am. J. Physiol. Lung Cell Mol. Physiol. 287, L52-59 (2004).
21. Chandramouli, S. et al. Tesk1 interacts with Spry2 to abrogate its inhibition of ERK phosphorylation downstream of receptor tyrosine kinase signaling. J. Biol. Chem. 283, 1679-1691 (2008).
22. Fong, C.W. et al. Tyrosine phosphorylation of Sprouty2 enhances its interaction with c-Cbl and is crucial for its function. J. Biol. Chem. 278, 33456-33464 (2003).
23. Li, X., Brunton, V.G., Burgar, H.R., Wheldon, L.M. & Heath, J.K. FRS2-dependent SRC activation is required for fibroblast growth factor receptor-induced phosphorylation of Sprouty and suppression of ERK activity. J. Cell Sci. 117, 6007-6017 (2004).
24. Yusoff, P. et al. Sprouty2 inhibits the Ras/MAP kinase pathway by inhibiting the activation of Raf. J. Biol. Chem. 277, 3195-3201 (2002).
25. Feng, Y.H. et al. MicroRNA-21-mediated regulation of Sprouty2 protein expression enhances the cytotoxic effect of 5-fluorouracil and metformin in colon cancer cells. Int. J. Mol. Med. 29, 920-926 (2012).
26. Feng, Y.H. et al. Deregulated expression of sprouty2 and microRNA-21 in human colon cancer: Correlation with the clinical stage of the disease. Cancer Biol. Ther. 11, 111-121 (2011).
27. Sayed, D. et al. MicroRNA-21 targets Sprouty2 and promotes cellular outgrowths. Mol. Biol. Cell 19, 3272-3282 (2008).
28. Barbachano, A. et al. SPROUTY-2 represses the epithelial phenotype of colon carcinoma cells via upregulation of ZEB1 mediated by ETS1 and miR-200/miR-150. Oncogene 35, 2991-3003 (2016).
29. Holgren, C. et al. Sprouty-2 controls c-Met expression and metastatic potential of colon cancer cells: sprouty/c-Met upregulation in human colonic adenocarcinomas. Oncogene 29, 5241-5253 (2010).
30. Ordóñez-Morán, P. et al. SPROUTY2 is a β-catenin and FOXO3a target gene indicative of poor prognosis in colon cancer. Oncogene 33, 1975-1985 (2014).
31. Zhang, Q., Shim, K., Wright, K., Jurkevich, A. & Khare, S. Atypical role of sprouty in p21 dependent inhibition of cell proliferation in colorectal cancer. Mol. Carcinog. 55, 1355-1368 (2016).
32. Zhang, Q. et al. Atypical role of sprouty in colorectal cancer: sprouty repression inhibits epithelial-mesenchymal transition. Oncogene 35, 3151-3162 (2016).
33. Rhodes, D.R. et al. ONCOMINE: a cancer microarray database and integrated data-mining platform. Neoplasia 6, 1-6 (2004).
34. Watanabe, T. et al. Differential gene expression signatures between colorectal cancers with and without KRAS mutations: crosstalk between the KRAS pathway and other signalling pathways. Eur. J. Cancer 47, 1946-1954 (2011).
35. Sutterluty, H. et al. Down-regulation of Sprouty2 in non-small cell lung cancer contributes to tumor malignancy via extracellular signal-regulated kinase pathway-dependent and -independent mechanisms. Mol. Cancer Res. 5, 509-520 (2007).
36. Brady, S.C. et al. Sprouty2 association with B-Raf is regulated by phosphorylation and kinase conformation. Cancer Res. 69, 6773-6781 (2009).
37. Tsuchida, N., Ryder, T. & Ohtsubo, E. Nucleotide sequence of the oncogene encoding the p21 transforming protein of Kirsten murine sarcoma virus. Science 217, 937-939 (1982).
38. Schubbert, S. et al. Germline KRAS mutations cause Noonan syndrome. Nat. Genet. 38, 331-336 (2006).
39. Niihori, T. et al. Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome. Nat. Genet. 38, 294-296 (2006).
40. Burmer, G.C. & Loeb, L.A. Mutations in the KRAS2 oncogene during progressive stages of human colon carcinoma. Proc. Natl. Acad. Sci. U. S. A. 86, 2403-2407 (1989).
41. Almoguera, C. et al. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 53, 549-554 (1988).
42. Tam, I.Y. et al. Distinct epidermal growth factor receptor and KRAS mutation patterns in non-small cell lung cancer patients with different tobacco exposure and clinicopathologic features. Clin. Cancer Res. 12, 1647-1653 (2006).
43. Vogelstein, B. & Kinzler, K.W. Cancer genes and the pathways they control. Nat. Med. 10, 789-799 (2004).
44. Rosty, C. et al. Colorectal carcinomas with KRAS mutation are associated with distinctive morphological and molecular features. Mod. Pathol. 26, 825-834 (2013).
45. Yamauchi, M. et al. Assessment of colorectal cancer molecular features along bowel subsites challenges the conception of distinct dichotomy of proximal versus distal colorectum. Gut 61, 847-854 (2012).
46. Lièvre, A. et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res. 66, 3992-3995 (2006).
47. Beganovic, S. Clinical significance of the KRAS mutation. Bosn. J. Basic Med. Sci. 9 Suppl 1, 17-20 (2009).
48. Lemmon, M.A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117-1134 (2010).
49. Song, S., Rosen, K.M. & Corfas, G. Biological function of nuclear receptor tyrosine kinase action. Cold Spring Harb. Perspect. Biol. 5 (2013).
50. Brand, T.M. et al. Nuclear EGFR as a molecular target in cancer. Radiother. Oncol. 108, 370-377 (2013).
51. Dekker, E., Tanis, P.J., Vleugels, J.L.A., Kasi, P.M. & Wallace, M.B. Colorectal cancer. Lancet 394, 1467-1480 (2019).
52. Hsu, H.-C. et al. Mutations of KRAS/NRAS/BRAF predict cetuximab resistance in metastatic colorectal cancer patients. Oncotarget 7, 22257-22270 (2016).
53. Jimeno, A., Messersmith, W.A., Hirsch, F.R., Franklin, W.A. & Eckhardt, S.G. KRAS mutations and susceptibility to cetuximab and panitumumab in colorectal cancer. Cancer J. 15, 110-113 (2009).
54. Chu, C.A. et al. MiR-338-5p promotes metastasis of colorectal cancer by inhibition of phosphatidylinositol 3-kinase, catalytic subunit type 3-mediated autophagy pathway. EBioMedicine 43, 270-281 (2019).
55. Kim, H.J. & Bar-Sagi, D. Modulation of signalling by Sprouty: a developing story. Nat. Rev. Mol. Cell Biol. 5, 441-450 (2004).
56. Lee, C.C. et al. Overexpression of sprouty 2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis. Oncogene 23, 5193-5202 (2004).
57. Ahmed, D. et al. Epigenetic and genetic features of 24 colon cancer cell lines. Oncogenesis 2, e71 (2013).
58. Zhang, Y. et al. The Role of the miR-21/SPRY2 Axis in Modulating Proangiogenic Factors, Epithelial Phenotypes, and Wound Healing in Corneal Epithelial Cells. Invest Ophthalmol Vis Sci 60, 3854-3862 (2019).
59. Tsavachidou, D. et al. SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant. Cancer Res. 64, 5556-5559 (2004).
60. Lao, D.H. et al. A Src homology 3-binding sequence on the C terminus of Sprouty2 is necessary for inhibition of the Ras/ERK pathway downstream of fibroblast growth factor receptor stimulation. J. Biol. Chem. 281, 29993-30000 (2006).
61. Thomas, S.J., Snowden, J.A., Zeidler, M.P. & Danson, S.J. The role of JAK/STAT signalling in the pathogenesis, prognosis and treatment of solid tumours. Br. J. Cancer 113, 365-371 (2015).
62. Liotta, L.A. & Clair, T. Cancer. Checkpoint for invasion. Nature 405, 287-288 (2000).
63. Walker, C., Mojares, E. & Del Rio Hernandez, A. Role of Extracellular Matrix in Development and Cancer Progression. Int. J. Mol. Sci. 19 (2018).
64. Lu, P., Takai, K., Weaver, V.M. & Werb, Z. Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harb. Perspect. Biol. 3 (2011).