autophagosome like structures were found within the RAD001 BEZ235 combination in comparison to tumors treated with placebo or either drug alone. While a few were up regulated in tumors, nearly half were considerably down regulated, two of which, Atg5 and Atg7, are cyst suppressors in liver. Analysis of current microarray data sets from HCC patients, having the signature of A Foretinib c-Met inhibitor versus B, showed that the altered appearance of autophagy genes is connected with those patients having an unhealthy prognosis, as revealed in the Kaplan Meier plot of these subsets of HCC patients. Although BEZ235 and RAD001 stimulate autophagy, a task for autophagy in controlling DEN induced HCC would not be in keeping with reports thatautophagy is mediated by 4E BP1, because BEZ235 alone triggers 4E BP1 dephosphorylation to almost the same extent as that of the drug combination. Recent studies argue that mTORC1 can specifically suppress autophagy by phosphorylating autophagy initiating kinase ULK1 at S757. Thus, we questioned whether BEZ235 and RAD001 synergize to the answer and ULK1 S757 dephosphorylation and whether these results were 4E BP1/2 dependent. To determine autophagy and 4E BP1/2 reliability, we took advantage of a glutathione S transferase described betaine homocysteine methyltransferase reporter, whose cleavage represents Extispicy a cargo based autophagy end-point, and human embryonic kidney 293 cells stably expressing either a nonsilencing short hairpin RNA, or an shRNA against 4E BP1/2. Withdrawal of serum and aminoacids from shNS cells generated dephosphorylation of ULK1, 4E BP1, and S6K1, with comparable results obtained for ULK1 and S6K1 in sh4E BP1/2 cells. However, basal levels of the GST BHMT fragment were indistinguishable in shNS versus sh4EBP1/2 cells, as was the degree of fragment accumulation brought on by serum and amino acid withdrawal. Therapy with 5 nM RAD001 induced S6K1 dephosphorylation but had little impact on 4E BP1 and natural product libraries ULK1 phosphorylation or on the accumulation of the GST BHMT fragment in cells, with equivalent results obtained for ULK1 and S6K1 in sh4E BP1/2 cells. The combination of the two drugs had a synergistic effect on the deposition of the GST BHMT fragment and ULK1 S757 dephosphorylation independent of 4E BP1/2 and S6K1. Ergo, the induction of autophagy may appear independently of S6K1 and 4E BP1/2. RAD001 and BEZ235 induce autophagy in liver tumors The results above raised the issue of whether autophagy may possibly more closely follow regression of DEN induced tumors than 4E BP1 dephosphorylation. We examined liver tumors for autophagosome development by transmission electron microscopy, to examine this. The TEM pictures unveiled double membrane vesicles indicative of autophagosomes, that have been studded with small particles resembling ribosomes, constant with autophagosomes being derived from the endoplasmic reticulum.