chemotherapy theSFNtoleranceinSFN-RHCCcells

 X.Zhang,etal. Cancer Letters 481 (2020) 32–44 Epithelial-mesenchymal transition (EMT) is one of the underlying exhibited enhanced CD47 and glycolysis. SSL6 significantly reversed mechanisms by which tumor acquire resistance to chemotherapy theSFNtoleranceinSFN-RHCCcells.Moreimportantly,SSL6andSFN [45,46].Long-termexposuretolow-doseSFNcaninducetumorcellsto synergistically suppressed xenograft HCC growthinvivo. develop EMT and enhance tumor invasion, metastasis and the re- It is reported that CD47 can enhance the proliferative, anti-apop- sistancetoSFN[47,48].OurresultsshowedthatSFNpromotedthekey totic, metastatic and adhesive activities of cancer cells, as well as molecule of EMT, includingE-CADHERIN,VIMENTINandZEB1, while function as a “don\'t eat me” signal to help tumor cells escape the inhibited N-CADHERIN. SSL6 reversed the upregulated E-CADHERIN phagocytosisofphagocyticcells.Inimmunecells,blockingthesignalof and down-regulated N-CADHERIN induced by SFN (Supplementary CD47 on the surface of T cells can activate the immune function of + Fig.4C).TogetherthesedatademonstratethatSSL6re-sensitizesSFN-R dendriticcellsandpromotetheclearanceoftumorcells[11].CD47 T − HCC cells. cells can secrete CD47-containing vesicles to activate CD47 T cells, and thereby stimulate endothelial cells to promote angiogenesis [10]. Therefore, blocking CD47 can theoretically improve the effect of anti- 3.7. SSL6andSFNsynergisticallyattenuateHCCgrowthinvivo tumor therapy. Superiortotraditionalmonoclonalantibodies,asabacteria-derived To validate the anti-tumor effect of SSL6 in vivo, we administered protein, SSL6 can be rapidly and extensively expressed and purified by SSL6 and SFN upon MHCC97H cells were transplanted in nude mice prokaryotic expression systems, which greatly reduces the production subcutaneously (Fig. 7A). Compared with the control group, there was costs [29]. Moreover, since it is not an antibody, its useinvivo will not no significant change in blood routine, liver and kidney function of induce ADCC and CDC effect, avoiding immunotoxicity, and has po- mice treated with SSL6 (Supplementary Fig. 5A). SSL6 alone showed a tentialapplicationprospectsfortumortherapy[29].Here,wefirstused trend to suppress the tumor growth, and remarkably attenuated the EscherichiacolitoprokaryoticallyexpressandpurifySSL6.Combination tumor growth with the synergism with SFN (Fig. 7B the reduction treatment of SFN with SSL6 revealed that SSL6 dose-dependently percentage of SSL6vs. control was 44.58%, p = 0.1065; the reduction blockedCD47signalingtopromoteSFN-inducedapoptosisinHCCcells. percentage of SFNvs. control was 73.51%, p = 0.0035; the reduction It is well known that metabolic reprogramming is one of the pro- percentage of SFN + SSL6 vs. control was 84.7###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-5.png####8%, p = 0.0016; the minent hallmarks of tumor cells, in which the level of glucose catabo- reductionpercentageofSFN+SSL6vs.SFNwas42 cck8 solubility.54%,p=0.0108) lism, i.e., aerobic glycolysis, also known as the Warburg effect, is sig- (Fig. 7B and C). Furthermore, CD47 levels were inhibited in SSL6- nificantly higher than oxidative phosphorylation [50,51]. High level of treated tumors (Fig. 7D). 

The mRNA expression of CD47 and key mo- glycolysisisanimportantsourceofATPintumorcells Protease Inhibitor Cocktail,whichpromotes lecules in glycolysis, includingHIF1A,HK2,PFKP andGLUT1 in tumor the proliferation, metastasis, drug tolerance and escape of apoptosis of tissueswereconsistentlyincreasedbySFNbutweredecreasedbySSL6. tumor by producing variety of intermediate products [52–55]. Moreover, SSL6 reversed SFN-upregulated CD47 and these glycolytic Literature has reported that when SFN is administrated to treat HCC genes (Supplementary Fig. 5B). Together our results delineate the ac- cells, SFN not only functions as a tyrosine kinase inhibitor, but also tionofSSL6onsensitizingHCCcellstoSFNbyblockingCD47-mediated promotes the ability of tumor cells to uptake glucose and up-regulates glycolysis (Fig. 8). the level of Warburg effect. In the case of inhibition of glycolysis by combinedadministrationof2DG,theanti-tumorabilityofSFNonHCC 4. Discussion cells was significantly up-regulated [56,57]. In HCC cells with potent tolerance toSFN,inhibition ofphosphofructokinase2 (PFK2)promotes SFN is the standard first-line targeted drug for advanced HCC, SFN sensitivity [58]. 

The above reports indicate that inhibiting glyco- whereas the low-sensitivity and acquired resistance limits its benefits lysis of HCC cells promotes SFN sensitivity. Indeed, we showed here for patients [49]. SFN promotes the glycolysis of HCC cells, which has thatSSL6inhibitedtheactivationofPI3K/Akt/HIF-1byblockingCD47, been acceptedas a critical mechanism for HCCtolerantto SFN [34].In therebyinhibiting glycolysisandenhancing thesensitivityof HCCcells the present study, we showed that SSL6 could bind to CD47 and block to SFN M3814 (nedisertib). the downstream PI3K/Akt/HIF-1 signal, which attenuated the SFN- The novelties and importance of this study include at least the fol- upregulated glycolysis of HCC cells. The inhibition of PI3K/Akt lowing 3 folds. Firstly, SSL6 is currently the only microbiota-derived pathway further suppressed CD47 expression. Consequently, the SFN- protein that binds CD47. We synthesized and purified recombinant induced HCC cell apoptosis was promoted, indicating that SSL6 sensi- SSL6 with cell-free protein synthesis method, using an Escherichia coli tized HCC cells to SFN. Furthermore, the SFN-tolerant HCC cells also Fig. 8. A schematic model of SSL6 sensitizes HCC cells to SFN by down-regulating glycolysis.