Autophagy Enhances The Release Of HMGB1 From Cancer-associated Fibroblasts To Stimulate The Stemness Of ER+ Breast Cancer Cells

Breast cancer（BRC）has been severely threatening the health and life of women.Recently,studies on cancer stem cell have provided new understanding of breast cancer therapy.It is demonstrated that cancer stem cells are powerful in self-renewal,proliferation,and differentiation.Meanwhile,cancer stem cells are located in the special microenvironment（niche）,which play important roles in maintaining and activing stemness.In breast cancer,CAFs composed tumour niche is indeed molecularly distinct from those found in normal stroma.Breast CSCs（BCSCs）were originally characterized based on the expression of aldehyde dehydrogenase 1（ALDH1）as a marker of BCSCs,capable of self-renewal,multilineage differentiation,and tumor initiation,reside in a niche comprised by cancer associated fibroblasts（CAFs）.CAFs are predominant components in breast cancer microenvironment and play significant roles in occurrence and progress of breast cancer.Co-culture with cancer cells revealed CAFs constitute a supporting niche for CSC pathway in breast cancer and lung cancer.Recently,autophagy is required for CSCs to support the survival and maintenance in cancers of breast,brain and colon.Except cancer cells,CAFs,vascular endothelial cells or other cells are also response to perturbation of tumor microenvironment,caused by inadequate supply of blood and nutrition induces stress.The state and mechanism of autophagy in CAFs still are unclear.We found CAFs in human breast cancer tissues show high level of autophagy and significantly correlated with ALDH1A1-positive breast cancer stem-like cells.In the general sense,autophagy means a lysosome-based cellular degradation system regulated by autophagy-related（ATG）gene products.Furthermore,recent evidence showed some leadless proteins need autophagic membranes for efficient enveloping and exocytosis.This unconventional secretion contributes to the exocytosis of nuclear protein high-mobility group box 1 protein（HMGB1）,which may be invoved in interation of CAFs and cancer cell.Here we report that in ER+breast cancer,CAFs expressing high levels of autophagy protein LC3II is associated with poor prognosis.LC3 II high-CAF/ER+breast cancer appears to be enriched with ALDH1-positive tumor initiating cells.Mechanistically,CAFs secrete HMGB1 to activate the receptor TLR4 expressed by ER+BCSCs to support their stemness.These findings indicate that the autophagy of CAFs in breast cancer niche plays a critical role in promoting the progression of ER+breast cancer.Main results and conclusions are as follows1.Correlation between autophagy activity in CAFs and poor prognosis and ALDH1A1 positive BCSCs in ER+breast cancer（1）IHC staining of LC3Ⅱin primary invasive ductal carcinoma（IDC）specimens obtained from a clinical cohort of 413 patients who underwent surgical resections.LC3Ⅱ,in primary invasive ductal carcinoma（IDC）specimens obtained from a clinical cohort of418 patients who underwent surgical resections.Based on the LC3Ⅱexpression by cancer cells and CAFs,the patient samples were designated as high-LC3ⅡCAFs,low-LC3ⅡCAF,high-LC3Ⅱcancer cells,low-LC3Ⅱcancer cells four groups.（2）Data analysis showed that in ER+breast cancer,high expression of LC3II in CAFs is positively correlated with the levels of ALDH1A1（P<0.05*）.（3）The statistical analysis showed a significant difference in Kaplan-Meier estimates of overall survival in ER⁺IDC based on LC3 B expression on CAFs.P values were obtained from two-sided Log-rank test.（4）A proportion of cancer replase in four groups was analyzed.The results showed high-LC3 B CAF groups have a high replase regardless of cancer cells in ER⁺IDC（P<0.05*）.2.BCSC enrichment and tumor progression promoted by autophagic CAFs in ER+breast cancer.（1）We isolated primary CAFs from ER⁺IDC patients without any presurgical therapy.On the quantity of autophagy gene levels,CAFs were divided into CAF1,CAF2 and CAF3,and CAF3 showed highest expression level of autophagy genes.We collected the cell supernatants from different CAFs stimulating autophagy with serum starvation as conditioned medium（CM）to culture ER⁺breast cancer cell line MCF-7 cells and ER^-breast cancer cell line MDA-MB-231 cells.Flow cytometry detection showed that ALDH1activity in MCF-7 cells is significantly elevated under CAF3-CM,which has high expression of Atg（P<0.05*）.（2）Conditioned medium from CAF3 significantly increased the mRNA expression of stem cell markers,OCT4,SOX2 and NANOG,and the sphere numbers of MCF-7.We knocked down a key autophagy gene ATG5,which were performed by transfection of specific shRNA targeting ATG5（sh ATG5）in CAF3.Significantly less ALDH1 activity with conditioned medium stimulation from CAFs3-shATG5 were observed compared with cells transfected with control sh RNA（shcontrol）,supporting that ATG5 is required for this mode in CAFs.Western blot analysis confirmed that the stemness marker,Oct4,Sox2 and Nanog in MCF-7 decreased under conditioned medium from sh ATG5-CAF3.（4）We further confirmed that the sphere-forming ability of MCF-7 cells cultured with conditioned medium of CAFs was reduced following the known-down of ATG5（P<0.05*）.Furthermore,co-injection of CAFs with MCF-7 cells markedly promoted the tumourigenesis in NOD-SCID mice and CAF3-shAtg5 abolished this effect（P<0.05*）.（5）In breast cancer,IHC double-staining of LC3 B and ALDH1A1 was detected in the same primary ER⁺IDC specimens.These results indicate that some secreted factors in CM from CAFs undergoing autophagy mediate the effect of CAFs on the self-renewal of BCSCs and tumorigenesis of ER+breast cancer.3.Release of HMGB1 by autophagic CAFs（1）In these experiments,we aimed to test the hypothesis that HMGB1,IL-18 and IL-1βwould be released upon CAFs autuphagy induced by serum starvation culture.CAFs with serum starvation led to release of HMGB1,but no IL-1βor IL-18 as detected by ELISA in the cell culture supernatants（P<0.01**）.（2）We further detected the supernatants from different CAFs and found HMGB1 in CAF3 is the most among three CAFs.（3）Immunofluorescence staining showed that HMGB1 abundantly located in the nucleus of CAFs,whereas after stimulating autophagy by serum starvation,HMGB1 predominantly exited the cell nucleus to the extracellular environment.（4）We examined HMGB1 and the key marker of autophagosomes LC3II by immunofluorescence confocal microscopy,HMGB1 and LC3II colocalized in CAFs stimulating autophagy by serum starvation.（5）Our data showed that knockdown of ATG5 in CAFs diminished the level of the secreted HMGB1 in supernatant and resulted in the accumulation of HMGB1 in cytoplasm after starvation culture for 48h,indicating HMGB1 extracellular release in an ATG5-dependent manner（P<0.05*）.4.Extra-HMGB1 was mainly from CAFs depended on non-classical autophagy.（1）The supernatants from MCF-7 cells and CAFs incubated with serum starvation were measured HMGB1 secretion by ELISA.The concentration of HMGB1 in CAF-CM was obviously higher than it in MCF-7-CM.（2）Western blot detection revealed increased LC3I to LC3II conversion and p62 degradation in MCF-7 cells under starvation.However,increased LC3I to LC3II conversion and p62 accumulation was observed in CAFs under starvation.Protein of p62 is selectively incorporated into autophagosomes through direct binding to LC3 and is efficiently degraded by autolysosome;thus,the total cellular expression levels of p62 inversely correlate with autophagosomes.（3）we transfected CAFs with RFP-GFP-LC3.After inducing autophagy,the autophagyosome was yellow,and autophagolysosome,which the lysosome contains an autophagosome,was red.Interestedly,CAFs different from MCF-7 cells to form red autophagolysosome maintained a large number of yellow autophagyosome stimulating autophagy by starvation for 48h.（4）Western blot showed that HMGB1 in CAFs cytoplasm decreased under starvation.At the same sample,the collected supernatants have significantly high levels of HMGB1.（5）Serial sections of ER⁺breast cancer sample were stained with antibodies against LC3II and HMGB1.The results showed that the section with low levels of LC3II in CAFs showed the location of HMGB1 in the nucleus and cytoplasm,however,with high levels of LC3II in CAFs,HMGB1 significantly diminished in the nucleus and cytoplasm,revealing that autophagy of CAFs enabled HMGB1 to secrete into the microenvironments.（6）when co-culturing MCF-7 cells with conditioned medium from MCF-7 cells and CAFs under starvation,flow cytometry indicated that ALDH1 activity significantly increased upon exposure to CAFs compared to MCF-7 cells.The results indicated that secretion of HMGB1 was derived from CAFs,but not MCF-7 cells in an autophagosome dependent manner.5.Requirement of Extra-HMGB1 for self-renewal of BCSCs（1）Treated with different concentration of HMGB1,MCF-7 cells showed higher m RNA levels of the stemness markers OCT4,SOX2 and NANOG in dose-dependent manne.Western blot analysis indicated that HMGB1（1μg/ml）promoted the expression of the stemness markers Oct4,Sox2 and Nanog.（2）Addition of HMGB1 also markedly elevated ALDH1 activity and sphere-forming ability of MCF-7 cells.（3）The flow cytometry showed that the self-renewal of MCF-7 cells was indeed dependent on release of HMGB1by CAFs,since its blockade with glcyrrhizic acid ammonium salt（GL）,which inhibits activities of HMGB1.（4）Combined MCF-7 cells with CAFs were injected into mammary fat pad with or without GL（10 mg/kg/day）.The results showed that GL significantly inhibited tumour growth.The number of ALDH1A1 positive cells in tumour with GL was markedly decreased to compare with tumour without it.These results confirm that HMGB1released by autophagic CAFs is critical for promoting the stemness of BCSCs.6.TLR4,HMGB1 receptor,involved in the interaction between CAFs and BCSCs.（1）Previous researches have demonstrated that the extracellular functions of HMGB1are mediated by binding to its endogenous receptors,such as the Receptor for Advanced GLcation End products（RAGE）as well as the Toll Like Receptors 2,4 and 9（TLR2,TLR4,and TLR9）.Pearson correlation analysis was used to determine the correlation coefficient（r²）between the ALDH1A1 and the receptors in TCGA database of ER⁺breast cancer.The levels of ALDH1A1 exhibited strong correlation（r²=0.5973,p<0.001）with TLR4.（2）When treating with LPS,the ALDH1 activity of MCF-7 cells significantly increased,suggesting that the TLR4 expressed by MCF-7 cells was functional and contributed to the stemness.（3）Furthermore,TLR4 antagonist,TAK-242,significantly diminished ALDH1activity induced by HMGB1.TAK-242 also abrogated the increased protein expression of the stemness markers Oct4,Sox2 and Nanog induced by HMGB1.（4）Sorted TLR4+MCF-7 cells have higher ability of sphere-formation than TLR4-MCF-7 cells.Moreover,CAF3-CM significantly elevated the spheres of TLR4+MCF-7 cells.Injection of TLR4+MCF-7 cells,but not TLR4-MCF-7 cells,into NOD-SCID mice,initiated tumors.Co-injection of autophagic CAF3 with TLR4+MCF-7 cells further enhanced the tumorigenicity.These results confirm the requirement of TLR4 on breast cancer cells to respond to the stimulating activity of HMGB1 released by autophagic CAFs.7.Correlation between LC3II/TLR4 levels and poor prognosis of ER+breast cancers（1）For examine the clinical relevance and importance of autophagy and TLR4 in the progression of ER+breast cancer,specimens were stained with antibodies against LC3II and TLR4 via IHC.The levels of LC3II in CAFs and TLR4 in cancer cells were scored to high or low LC3II and TLR4 protein expression.In 303 ER+breast cancer cases,145（47.9%）were TLR4^high cancer cells and 158（52.1%）were LC3II^high CAFs.（2）The patients with high-level of LC3II in CAFs demonstrated significantly poorer overall survival and relapse-free survival（P=0.018*;P=0.021*）.High level of TLR4 in cancer cells demonstrated poorer overall survival and significantly poorer relapse-free survival（P=0.060;P=0.025*）.TCGA database analysis showed that TLR4 in cancer cells is significantly poorer overall survival than low-level expression in a larger sample size（n=604;P<0.05*）.（3）Combined detection of LC3II and TLR4 revealed that patients with LC3II high in CAFs and TLR4 high in cancer cells demonstrated significantly poorer overall than those with LC3-II low in CAFs low and TLR4 low in cancer cells.Cox proportional hazards regression analysis further demonstrate that combined detection of LC3II and TLR4 provide a novel prognostic index for predicting overall survival（HR=7.059,95%CI=1.126-44.245;P=0.037*）and relapse-free survival（HR=2.735,95%CI=1.111-6.6.734;P=0.029*）in ER+breast cancer patients.