Investigation of anticancer activity of the natural compound halofuginone for modulating autophagic flux through phosphorylation of ULK1 in colorectal cancer

Autophagy is a key process in the development of cancer. Our previous study found that halofuginone (HF), a natural compound isolated from the traditional Chinese herbal plant Dichroa febrifuga Lour., exerted anticolorectal cancer (CRC) activity both in vitro and in vivo. Interestingly, HF downregulated mammalian target of rapamycin complex 1 (mTORC1) and decreased phosphorylation of ULK1 at Ser757 with enhanced autophagic marker LC3-II expression in CRC cells in a dose-dependent manner, indicating HF may induce autophagy under nutrient-rich condition. On the other hand, HF upregulated mTORC1 and phosphorylation of ULK1 at Ser757 as well as significantly reduced LC3-II expression under serum deprivation, suggesting that HF may inhibit autophagy. Therefore, we hypothesize that HF may exert anticancer activity through regulating one key process of autophagy, phosphorylation of ULK1, to either induce autophagy under nutrient-rich condition or inhibit autophagy under starvation conditions. To test this hypothesis, firstly, phosphorylation of ULK1 at Ser757 regulated by mTORC1 or at Ser317/Ser777 regulated by AMP-activated protein kinase, will be systematically investigated under nutrient-rich or starvation conditions (glucose starvation and serum deprivation in this proposed study). More importantly, ULK1/mAtg13/FIP200 complex encoded by ULK1 and two subunits Atg13 and FIP200 which signal to autophagy regulators will be examined with HF treatment. Secondly, in order to understand the dynamic changes of autophagy upon HF treatment under nutrient-rich condition, autophagic flux analysis including lysosomal enzyme cathepsin activity and co- staining of GFP-LC3 with LysoTracker Red or LAMP1 antibody for autophagosome-lysosome fusion detection will be carried out in time- and dose- dependent manners in CRC cell lines. Also, autophagy inhibitors chloroquine and bafilomycin A1 will be used to validate the autophagic flux induced by HF. Thirdly, considering that nutrient-stress can induce autophagy for cancer cell survival, HF will further be used in CRC cell lines under starvation conditions to investigate whether and how HF inhibits autophagic flux, in which mTORC1 activation has been observed in cancer cells upon HF treatment. Fourthly, in order to mimic the nutrient-stress condition in vitro, we will further investigate whether HF exerts anticancer activity under caloric restriction in vivo by using human CRC tumor xenografts in nude mice. The results will be compared with our previous studies with standard chow diet. Based on these studies, we would shed light on autophagic mechanisms regulated by phosphorylation of ULK1 upon HF treatment and provide new evidence of HF as a novel autophagic modulator for the potential therapy in CRC.