Lactate Inhibits ATP6V0D2 Expression In Tumor-associated Macrophages To Promote HIF-2?-mediated Tumor Progression

Objective: Tumor-associated macrophages present with the highest proportion among tumor infiltrating immune cells,and their phenotype and function are regulated by tumor microenvironment clues from tumor cells or stromal cells.Tumor-associated macrophage can promote tumor progression via a variety of mechanisms.Moreover,most tumor cells utilize glycolysis to metabolize glucose to produce lactate within the tumor,and lactate can regulate various signaling pathway or directly act as a signaling molecule to regulate cells growth and differentiation.However,the mechanism of how tumor-derived lactate affects the phenotype and function of tumor-associated macrophage to promote tumor progression is not fully understood.We used tumor-derived conditional medium to stimulate macrophage and found that the expression of lysosome protein ATP6V0d2 in macrophage was significantly inhibited.Therefore,our current study explores the molecule mechanism of the regulation of ATP6V0d2 on tumor-associated macrophage by tumor metabolite lactate and its effect on tumor growth.Methods: 1.Real-time PCR analysis of the expression of macrophage-associated lysosomal gene in BMDMs stimulated with(LLC-TCM)and (B16-F10-TCM).To confirm the down-regulation of Atp6v0d2 was associated with lactate from tumor cells,and explore the relevant signaling pathways by flow and Western Blot analysis.2.The specific knockout mice have been constructed successfully,LLC and B16-F10 tumor cells were subcutaneously inoculated WT mice and knockout mice,observed and recorded the tumor growth rate.At day 15,the mice were sacrificed,and tumor tissues were isolated and tumor size and weight were compared between WT and Atp6v0d2 mice.Western blot analyzed the expression of ATP6V0d2 in tumor cells,lymphocytes,fibroblasts and macrophages.To confirm that the effect of Atp6v0d2 deletion on tumor cell growth was intrinsic to TAM function,Atp6v0d2-/-mice were injected s.c.with 5×105 LLC cells alone or 5×105 LLC cells plus either 2×105 WT BMDMS or Atp6v0d2-/-BMDMs,or injected s.c.with 5×105 B16-F10 cells alone or 2×105 B16-F10 cells plus either 2×105 WT BMDMs or Atp6v0d2-/-BMDMs.Tumor growth and weight were monitored.3.The mouse lung adenocarcinoma cell line LLC(8×105)was inoculated into the WT and Atp6v0d2-/-mice.At day 16,the mice were sacrificed.The tumor nodules were observed,and the lung tissues of these groups were isolated and digested into single cell suspensions.Flow cytometry analysis of macrophage polarization and tumor cell proliferation and apoptosis in WT mice and Atp6v0d2-/-mice.4.In the subcutaneous tumor model of WT mice and Atp6v0d2-/-mice,flow cytometry analysis of the tumor infiltrating macrophage polarization in WT mice and Atp6v0d2 knockout mice.The tumor tissues were digested into single cell suspensions and then subjected to CD11 b magnetic,real-time PCR analysis of the expression of Mrc-1,Arg-1,Fizz-1,IL-1? and Il-6 in tumor tissues or isolated with CD11 b magnetic beads from WT mice and Atp6v0d2-/-mice.WT and Atp6v0d2-/-BMDMs were stimulated with medium or LLC-TCM for 6h or 36 h.The expression of Il-1b and Il-6or Arg-1 and Fizz-1 was determined by q-PCR.5.In the subcutaneous tumor model,double immunofluorescence staining(CD31 and ?-SMA)vessels in tumor tissues from WT mice and Atp6v0d2-/-mice;HE staining on the tumor tissues in WT mice and Atp6v0d2-/-,compared the percentage of hemorrhagic area versus the whole tumor area;ELISA further detected the production of vascular endothelial growth factor(VGEF)in tumor tissue from WT mice and Atp6v0d2-/-mice;real-time PCR analysis the expression of Vegf in tumor tissues and isolated with CD11 b magnetic beads from WT mice and Atp6v0d2-/-.WT and Atp6v0d2-/-macrophages were stimulated with LLC-TCM and B16-F10-TCM for 6 h in vitro,and Vegfexpression was detected by real-PCR.6.Whole-mount immunofluorescence staining(HIF-2? and F4/80)in tumor tissue of WT and Atp6v0d2-/-mice,since HIF-1? is also a very important transcription factor that regulates angiogenesis,in order to exclude HIF-1? function,WT and Atp6v0d2-/-macrophages were stimulated by LLC-TCM in both normoxia and hypoxic conditions for the different time points(0,2,4,6 h)respectively,western Blot detected the expression of HIF-1?,HIF-2?,P62 and LC3 proteins.Furthermore,WT BMDMs were transduced with control virus or retroviral ATP6V0d2 virus,followed with treatment of LLC-TCMfor the indicated times.The expression of HIF-2?,P62,ATP6V0d2,and LC3 was determined by immunoblottingunder normoxia conditions in vitro.To further exclude the effects of proteasome,WT and Atp6v0d2-/-BMDMs were treated with proteasome inhibitor(MG132)at different time points.(0,2,4,6h),immunoblotting of HIF-2?,P62 and LC3 protein;Lysosomal inhibitor(Baf A)treatment of WT and Atp6v0d2-/-BMDMs for 6h,western Blot detection of HIF-2?,P62 and LC3 protein;at the same time,MG132 treatment of WT and Atp6v0d2-/-BMDMs for 4h,immunofluorescence analysis of the colocalization of HIF-2? and Lys-tracker in WT and Atp6v0d2-/-BMDMs.7.For the inhibition of HIF-2? in a mouse model: a total of 5×105 LLC cells in a volume of 100 ?l of PBS were inoculated s.c.into 6-7 weeks of age C57BL/6 WT or Atp6v0d2-/-male mice,and then HIF-2?-specific inhibitor(PT2385)was used to abrogate HIF-2?-regulated tumor growth.Once tumor size reached 100-200 mm3 at day 10,WT and Atp6v0d2-/-mice were treated with PBS or PT2385(5mg/kg)twice daily by oral gavage for 4 consecutively days.After the 15 th day,the mice were sacrificed,the tumor tissues were isolated,and the tumor size and weight of each group were compared.Immunofluorescence staining(CD31 and ?-SMA)of tumor tissues from each groups;real-time PCR analysis of the expression of Mrc-1,Arg-1,Fizz-1 and Ym-1 in tumor tissues of each group;in order to further verify the regulation of tumor macrophage polarization by HIF-2?,WT and Atp6v0d2-/-macrophages were stimulated with(LLC-TCM)for 24 h with or without PT2385 and PT2385 was added 18 h prior to the TCM stimulation in vitro,real-time PCR analysis the expression of Mrc-1,Arg-1 and Fizz-1in WT and Atp6v0d2-/-mice.8.To investigate the significance of the ATP6V0d2-mdeiated HIF-2? lysosomal degradation in human cancers: immunofluorescence was used to detect the colocalization of HIF-2? and CD68 in human lung adenocarcinoma;HE and histochemical staining of precancerous tissues and cancer tissues of lung cancer patients,analysis and comparison of CD163,ATP6V0d2 and HIF-2? correlation;according to the ATP6V0d2 expression level,lung cancer patients were divided into high expression of ATP6V0d2 group and low expression of ATP6V0d2 group,comparison of the ATP6V0d2 and HIF-2?expression and survival time;statistical analysis of the relationship between the expression of ATP6V0d2 and the number of polarized macrophages in tumor tissues.Results: 1)LLC-derived TCM enhanced the expression of multiple V-ATPase subunits,the expression of Atp6v0d2 was significantly reduced in the presence of TCM,and macrophages stimulated with different ratios of LLC-TCM:complete medium and found that at a ratio of 1:3 or above,TCM strongly suppressed the expression of Atp6v0d2.In addition,boiled TCM(b-TCM)derived from LLC cells,as well as from B16-F10 cells,still inhibited the Atp6v0d2 in macrophages,suggesting that the active agent in the TCM was heat-resistant and likely to be a low molecular weight metabolite.Lactic acid is a well-known product of tumor cell glycolysis.We found the lactate concentration was around 34.5m M in LLC-TCM and 45.2m M in B16-F10-TCM.To test the effect of lactate on Atp6v0d2 expression,we incubated macrophages with media containing varying concentrations of lactate and found that lactate inhibited Atp6v0d2 expression in a dose-dependent mannerover a range of 2m M to 50 m M.To confirm that the inhibition of Atp6v0d2 was due to the presence of lactate in the TCM,we used various concentrations of monocarboxylic acid transport inhibitor 2-Cyano-3-(4-hydroxyphenyl)-2-propenoic acid(CHC)in macrophages to block lactate transport.Addition of CHC(5m M)almost completely reversed the inhibition of Atp6v0d2 expression by LLC.Atp6v0d2 is a target gene of TFEB,a master regulator of lysosomal biogenesis,LLC-TCM and boiled LLC-TCM,as well as lactate,induced ribosomal protein S6 phosphorylation,a surrogate marker of m TORC1 activation.This was associated with significantly reduced nuclear localization of TFEBupon lactate or TCM treatment,using Ch IP-q PCR,we found that TFEB directly bound to the Atp6v0d2 promoterand the binding of TFEB was significantly reduced by the addition of lactateor TCM.Furthermore,addition of the m TOR inhibitor Torin reversed the down-regulation of Atp6v0d2 expression by lactate or TCM.2)Deletion of Atp6v0d2 led to significantly increased tumor size,compared with WT mice,we also obtained the similar results with B16-F10 melanoma.As to Atp6v0d2 is expressed in BMDM and TAM but not in CD4+,CD8+ T cells and other tumor cells,the co-inject experiment showed the effect of Atp6v0d2 deletion on tumor cell growth was intrinsic to TAM function.3)In the lung metastasis experiment,Atp6v0d2-/-mice exhibited significantly more tumor nodules and higher proportion of protumoral CD11b+F4/80+CD206+,compared with those from WT mice.Furthermore,the metastasized tumor cells(gated on CD45-)isolated from the lungs of Atp6v0d2-/-mice expressed higher amounts of Ki-67.In addition,the tumor cells from the lungs of Atp6v0d2-/-mice were less apoptotic.4)In the model of LLC-induced tumor,deletion of Atp6v0d2 resulted in an increased percentage of CD206+CD11C-protumoral cells within the F4/80+ macrophage compartment but had no effect on the percentage of CD206-CD11C+ pro-inflammatory macrophages.Furthermore,the expression of protumoral macrophage associated factors such as Mrc1,Arg1 and Fizz1 withinthe tumor tissues and isolated TAMs were significantly higher in Atp6v0d2-/-tumor bearing mice compared with tumors in control animals,but pro-inflammatory-associated Il1 b and Il6 expression was similar.In addition,LLC TCM induced significantly higher expression of Arg1 and Fizz1 in Atp6v0d2-/-BMDMs,compared with wild type BMDMs.By contrast,the absence of ATP6V0d2 resulted in reduced expression of Il-1b but not Il-6 in macrophages upon treatment of LLC TCM,compared with wild type BMDMs.5)In the LLC-induced tumor model,Atp6v0d2-/-mice had increased vascularizationand reduced mature vessel generation as measured by CD31 and ?-SMA stainingrespectively,together with enhanced hemorrhages.Meanwhile,the level of tumor VEGF was significantly enhanced within the tumors that grew in the Atp6v0d2-/-mice;the expression of Vegf was enhanced in tumors and isolated TAMs in the Atp6v0d2-/-mouse group.The addition of LLC-and B16-F10-TCM led to enhanced Vegfexpression after 6 h that was significantly greater in the Atp6v0d2-/-BMDMs,compared to WT controls.6)Tumor tissue immunofluorescence revealed that HIF-2? expression was increased in the Atp6v0d2-deficient TAMs,suggesting post-translational regulation of HIF-2? by ATP6V0d2.A series of western blot experiment in vitro showed TCM induced HIF-2? accumulationwhile suppressed ATP6V0d2 expression in WT macrophages;the expression levels of HIF-2? were higher after 2h,4h,and 6h TCM stimulation in the absence of ATP6V0d2,accompanied with enhanced autophagy-associated protein expression of P62 and LC3.By contrast,deletion of ATP6V0d2 did not increase HIF-1? protein concentration upon TCM stimulation.Hypoxia stimulation of 4h induced significant accumulation of HIF-1?,as well as HIF-2?.However,deletion of ATP6V0d2 resulted in enhanced HIF-2? but not HIF-1? expression,indicating that the specific regulation of HIF-2? by ATP6V0d2 is oxygen-independent.We found that treatment of MG132 led to an accumulation of HIF-2? at 2h and persisted until 6h in the WT cells,indicating a proteasome-mediated degradation of HIF-2?,however,HIF-2? protein concentration remained significantly higher in Atp6v0d2-deficeint cells,compared with WT control in the presence of MG132,suggesting a second proteasome-independent regulation of HIF-2? degradation by ATP6V0d2.Macrophages incubated in the presence of Bafilomycin A resulted in enhanced HIF-2? in wild type cells but not in Atp6v0d2-/-cells and the expressions of ATP6V0d2 and p62 were comparable after Bafilomycin treatment.Treatment of macrophages with MG132 to block the proteasome degradation pathway resulted in enhanced co-localization of HIF-2? and Lyso Tracker in wild type cells whereas the co-localization of HIF-2? and Lyso Tracker was significantly reduced in the Atp6v0d2-/-cells.7)The specific inhibitor of HIF-2?(PT2385)can significantly inhibit growth of subcutaneous tumor in WT and Atp6v0d2-/-mice,and also can reduce the formation of abnormal blood vessels.Furthermore,the expression of macrophage genes associated with a protumoral phenotype including Mrc1,Arg1,Fizz1,and Ym1 in tumors isolated from Atp6v0d2-/-mice was again reversed by PT2385.Treatment of WT and Atp6v0d2-/-BMDMs stimulated with LLC-TCM with PT2385,that resulted in a significantly reduction in the expression of Arg1,Fizz1 and Ym1 in WT and Atp6v0d2-/-BMDMs and eliminated the differences between the two groups,which is consistent with the results of the above tumor models.8)In the clinical lung adenocarcinoma patients,HIF-2? was mainly expressed in the CD68+ macrophages,there were more CD163+protumoral-like macrophages and enhanced HIF-2? in the ATP6V0d2 low expression group.In addition,we found the survival time of patients for with high ATP6V0d2 was significantly extended compared with the patients that had low ATP6V0d2 expression,and the patients with higher expression levels of HIF-2? had a significantly shorter survival time than the patients with lower level of HIF-2?.Conclusion:In the tumor microenvironment,tumor cell-derived lactate can suppress Atp6v0d2 expression in macrophage via activation of m TORC1 that lead to reduced lysosomal degradation of HIF-2?.Subsequently,macrophages produced more VEGF and were polarized into a protumoral phenotype by lactate,which in turn to promote tumor progression.We found that tumors grew faster in Atp6v0d2-deficient mice compared with control animals.Meanwhile,in the clinical specimens of lung adenocarcinoma,the expression levelof ATP6V0d2 and HIF-2? in TAMs correlated positively and negatively with patient survival respectively,highlighting the significance of the regulation of HIF-2? by ATP6V0d2 in human cancers.