Inhibition of VEGF induces cellular senescence in colorectal cancer cells Mohammad R. Hasan 1,2 , Shirley H. Y. Ho 1,2 , David A. Owen 3 and Isabella T. Tai 1,2 1 Division of Gastroenterology, Department of Medicine, University of British Columbia, Vancouver, BC, Canada 2 Genome Sciences Centre, BCCA, Vancouver, BC, Canada 3 Department of Pathology, University of British Columbia, Vancouver, BC, Canada Vascular endothelial growth factor (VEGF) inhibitors, such as bevacizumab, have improved outcomes in metastatic colorectal cancer (CRC). Recent studies have suggested that VEGF can delay the onset of cellular senescence in human endothelial cells. As VEGF receptors are known to be upregulated in CRC, we hypothesized that VEGF inhibition may directly influence cellular senescence in this disease. In our study, we observed that treatment with bevacizumab caused a significant increase (p < 0.05) in cellular senescence in vitro in several CRC cells, such as MIP101, RKO, SW620 and SW480 cells, compared to untreated or human IgG-treated control cells. Similar results were also obtained from cells treated with a VEGFR2 kinase inhibitor Ki8751. In vivo, cellular senescence was detected in MIP101 tumor xenografts from 75% of mice treated with bevacizumab, while cellular senescence was undetectable in xenografts from mice treated with saline or human IgG (p < 0.05). Interestingly, we also observed that the proportion of senescent cells in colon cancer tissues obtained from patients treated with bevacizumab was 4.4-fold higher (p < 0.01) than those of untreated patients. To understand how VEGF inhibitors may regulate cellular senescence, we noted that among the two important regulators of senescent growth arrest of tumor cells, bevacizumab-associated increase in cellular senescence coincided with an upregulation of p16 but appeared to be independent of p53. siRNA silencing of p16 gene in MIP101 cells suppressed bevacizumab-induced cellular senescence, while silencing of p53 had no effect. These findings demonstrate a novel antitumor activity of VEGF inhibitors in CRC, involving p16. Colorectal cancer (CRC) as one of the most common forms of cancer, accounted for 639,000 deaths worldwide in 2009 (WHO, 2009). At present, mortality rates still remain high due to the development of chemotherapy resistance, espe- cially to drugs used in conventional first-line therapy, such as 5-flourouracil (5FU), a DNA synthesis inhibitor and irinote- can (CPT-11), a topoisomerase inhibitor. 1 Fortunately, the introduction of bevacizumab (AvastinV R ) for second-line treatment of metastatic disease, when administered in combi- nation with FOLFOX4 (5FU, leucovorin and oxaliplatin) 2 has improved the overall survival of patients with metastatic dis- ease. 3 Bevacizumab is a recombinant humanized monoclonal IgG1 antibody that binds and interferes with vascular endo- thelial growth factor (VEGF), inhibiting its biological effects. 4 Unlike 5FU or CPT-11, which target tumors directly, bevaci- zumab acts by indirectly reducing tumor progression by in- hibiting angiogenesis, 5 based on two proposed mechanisms: (a) by reducing tumor vasculature, which causes deprivation of nutrients and growth signals; and (b) by reducing vascular permeability in existing vessels, thus enhancing chemothera- peutic delivery. VEGF is a key regulator of angiogenesis: in vascular endo- thelial cells, stimulation by VEGF results in increased perme- ability, proliferation, migration and invasion. 6 Its receptor, VEGFR, is not only expressed in endothelial cells 7 but also it has been seen in breast, 8 bladder 9 and pancreatic 10 cancer cell lines. In addition, an immunohistochemical screening of non- endothelial cancer specimens revealed detectable levels of VEGFR in bladder, breast, intestinal and lung cancers. 11 Sev- eral CRC cell lines, including HT29, SW480, SW620 and RKO, have demonstrated VEGFR expression 12 and immunohisto- chemical analysis of clinical specimens also revealed that both VEGF and VEGFR are highly expressed in CRC. 13,14 These observations suggest a possible autocrine signaling pathway for VEGF within epithelial cells. In fact, inhibition of VEGF via siRNA can reduce cellular proliferation in RKO cells. 15 In addition to its effect on angiogenesis, studies of human dermal microvascular endothelial cells (HDMECs) exposed to VEGF 16 suggest that it may also delay the onset of Key words: VEGF-inhibition, cellular senescence, p16, p53, colorectal cancer Grant sponsor: Cancer Research Society, The Michael Smith Foundation for Health Research (MSFHR) and the Canadian Institutes of Health Research (CIHR); Grant number: MOP 82881 DOI: 10.1002/ijc.26179 History: Received 10 Jan 2011; Accepted 2 May 2011; Online 26 May 2011 Correspondence to: Isabella T. Tai, Division of Gastroenterology, Department of Medicine, University of British Columbia, 2775 Laurel Street, Vancouver, British Columbia, Canada V5Z 1M9, Tel.: 604-675-8150, Fax: þ604-675-8178, E-mail: itai@bcgsc.ca Cancer Cell Biology Int. J. Cancer: 129, 2115–2123 (2011) V C 2011 UICC International Journal of Cancer IJC