The Role Of Anastasis In Post-Chemotherapy Breast Cancer Relapse And Metastasis And The Underlying Mechanisms

Research background and aimBreast cancer is one of the most common malignancies in women worldwide.Among all cancers in women,breast cancer ranks the first in incidence and the second in mortality.Chemotherapy is a commonly-used therapy for breast cancer.Clinical application of chemotherapy dramatically improves the survival of breast cancer patients.However,cancer recurrence and metastasis after chemotherapy are the main reasons for poor prognosis.The mechanism underlying post-therapy recurrence and metastasis is complex and not fully understood.Elucidating the mechanism will help develop new therapeutics and improve survival of breast cancer patients.Anastasis is a process by which cells survive executioner caspase activation after transient exposure to apoptotic stress.Apoptosis induction is an important strategy to eliminate cancer cells by chemotherapy.Evading apoptotic cell death may be a major cause for cancer recurrence and metastasis.In recent years,it has been reported that some types of cancer cells can survive through anastasis after exposure to apoptotic stress including chemotherapy.Cancer cells that have undergone anastasis（anastatic cells）exhibit enhanced migration and drug resistance.However,the role of anastasis in breast cancer recurrence and metastasis after chemotherapy are not clear.The goal of this study is to investigate the role of anastasis in the post-chemotherapy recurrence and metastasis of breast cancer and the underlying mechanism.Methods and resultsPart Ⅰ The effect of anastasis on the malignant behaviors of breast cancer cells1.Breast cancer cells can survive through anastasis after treatment with chemotherapeutic drugs.In this study we adopted the m^CasExpress lineage tracing system to label cells that survived executioner caspase activation and their progeny with ZsGreen.To investigate whether breast cancer cells can undergo anastasis after treatment with chemotherapeutic drugs,we delivered m^CasExpress into two breast cancer cell lines to generate BT474Cac cells and MDA-MB-231^Cas cells and treated them with two commonly-used chemotherapeutic drugs adriamycin（ADR）and cisplatin（CDDP）.Using fluorescent microscopy,live imaging and flow cytometry,we found that some BT474^Cas cells and MDA-MB-231^Cas cells survived executioner caspase activation and proliferated after ADR or CDDP treatment,indicating that breast cancer cells can undergo anastasis after treatment with chemotherapeutic drugs.2.Recurrent breast cancer tissues after chemotherapy contain more anastatic cells.To determine whether anastasis participates in recurrence after chemotherapy,we injected MDA-MB-231^Cas cells into the fat pads of the nude mice and randomly allocated the mice into ADR（7.5mg/kg）treatment group or control group when the xenografts grew to around 50mm3.After 4-week treatment,the xenografts were no longer palpable in ADR treatment group,thus we stopped the chemotherapy and monitored the regrowth of the xenografts.Using flow cytometry,we analyzed the percentage of ZsGreen⁺cells in the xenografts from the treated group after recurrence and the control group.We found that xenografts from both groups contained ZsGreen⁺cells,but the percentage was significantly higher in the recurrent xenografts.3.The effects of anastasis on the malignant behaviors of breast cancer cells To investigate the phenotypic changes after anastasis,we sorted out and collected the ZsGreen⁺ cells（anastatic cells）and the ZsGreen^-cells（cells without executioner caspase activation）from BT474^Cas cells and MDA-MB-231^Cas cells recovered from ADR or CDDP treatment through fluorescence-activating cell sorting（FACS）.Cells treated with DOX alone were also applied to FACS and the ZsGreen" cells were collected as control populations.We then analyzed the phenotypes of the control,the ZsGreen^-,and the ZsGreen⁺cells derived from BT474^Cas cells and MDA-MB-231^Cas cells recovered from ADR or CDDP treatment,and obtained the following results.1)Anastatic breast cancer cells exhibited faster proliferation.Using EdU incorporation assays and colony formation assays,we found that the ZsGreen⁺cells proliferated faster than the ZsGreen" counterparts and the control cells.2)Anastatic breast cancer cells showed enhanced migration and invasion.Using transwell assays,we found that the migration and invasion were elevated in the ZsGreen⁺cells compared to those in the ZsGreen’ cells and the control cells.3)Anastasis promotes xenograft growth.We injected the ZsGreen⁺,the ZsGreen"and the control cells into the fat pads of the nude mice and found that compared to the xenografts formed by the ZsGreen" cells and the control cells,those formed by ZsGreen⁺cells grew faster and contained higher percentage of Ki67+cells.4)Anastasis promotes metastasis in vivo.To investigate the effect on tumor metastasis,we injected the ZsGreen⁺,the ZsGreen^-and the control cells intravenously through tail into nude mice and found that mice injected with the ZsGreen⁺cells contained more metastatic nodules in the lungs than mice injected with the other two groups of cells.4.Anastatic breast cancer cells exhibited elevated expression of CDH12.To figure out the molecular mechanism underlying the enhanced malignancy after anastasis,we performed RNA sequencing on the ADR-ZsGreen⁺and ADR-ZsGreen^-cells derived from BT474^Cas cells and MDA-MB-231^Cas cells recovered from ADR treatment and selected genes that were upregulated more than 4 folds in both ADR-ZsGreen⁺cells.We then assessed the expression of these genes in the control,the ZsGreen^-and the ZsGreen⁺cells derived from BT474^Cas cells and MDA-MB-231Cac cells recovered from ADR treatment or CDDP treatment using RT-qPCR.We found that only GDAP1L1 and CDH12 were significantly upregulated in all the ZsGreen⁺cells.CDH12 has been previously reported involved in growth and metastasis in salivary adenoid cystic carcinoma and colorectal cancer.Part Ⅱ CDH12 regulates the malignant behaviors of anastatic breast cancer cells through ERK/CREB1.Anastatic cancer cells increase proliferation and migration through upregulating CDH12 expression.1)The enhanced proliferation and metastasis in anastatic breast cancer cells rely on upregulation of CDH12.To determine the role of CDH12 in the anastasis-induced phenotypic changes,we knocked down CDH12 in the control,the ZsGreen^-and the ZsGreen⁺cells.We analyzed the proliferation using EdU incorporation assays and colony formation assays and the migration using transwell assays.It turned out that knocking down CDH12 suppressed proliferation and migration in all three groups of cells and attenuated the difference between them.Moreover,overexpression of CDH12 in BT474 and MDA-MB-231 cells increased proliferation and migration,suggesting CDH12 as a positive regulator of proliferation and migration in breast cancer cells.2)Knocking down CDH12 inhibited anastasis-enhanced xenograft growth and metastasis in vivo.We evaluated the effect of CDH12 knockdown on the capacity of breast cancer cells to grow and metastasize in nude mice and found that reducing CDH12 expression suppressed the growth of xenografts formed by anastatic cells and the lung metastasis of anastatic cells.3)Upregulation of CDH12 was associated with poor prognosis in breast cancer patients.Analysis on publicly available datasets revealed that CDH12 expression in breast cancer patients was associated with the stage and the size of the tumors and the poor prognosis.2.The mechanism underlying CDH12 regulation of cell proliferation and metastasis.1)CDH12 promotes proliferation and migration through activating CREB.Western blotting revealed increased phosphorylation of CREB in the anastatic cells,which was dramatically suppressed by knocking down CDH12.Both inhibition of CREB activity and interfering CREB expression significantly suppressed the enhanced proliferation and migration in anastatic cells.Overexpression of CDH12 in BT474 and MDA-MB-231 cells elevated CREB phosphorylation.Inhibition of CREB prevented elevation in proliferation and migration induced by CDH12 overexpression.2)CDH12 regulates CREB activity through ERK.To determine the protein that mediates CDH12 regulation of CREB,we analyzed the expression and phosphorylation of PKA,PKC and ERK,which were reported upstream of CREB.Only ERK phosphorylation was upregulated in all the ZsGreen⁺cells.Knocking down CDH12 reduced ERK phosphorylation.Inhibition of ERK activity or knocking down ERK rescued the increased CREB phosphorylation and enhanced proliferation and migration in the anastatic cells.Furthermore,overexpression of CDH12 significantly upregulated ERK phosphorylation in BT474 and MDA-MB-231 cells.Inhibition of ERK activity suppressed the increased CREB phosphorylation and enhanced proliferation and migration induced by CDH12 overexpression.3.Anastasis reduces the repressive epigenetic modifications in CDH12 promoter region.To investigate mechanism underlying CDH12 upregulation in anastatic cells,we analyzed DNA methylation in CDH12 promoter in the control,the ZsGreen^-and the ZsGreen⁺cells.We found that DNA methylation in CDH12 promoter was significantly reduced in the anastatic cells compared to that in the other two groups.qChIP assays revealed that the enrichment of the repressive histone modifications like H3K9me3,H3K27me3 and H2AK119ubl in CDH12 promoter was significantly reduced and the enrichment of the marker for transcription activation H3K4me3 was significantly increased.These data suggest the upregulated CDH12 expression may attribute to the epigenetic changes in CDH12 promoter.ConclusionsIn this study,we used two breast cancer cell lines and two commonly-used chemotherapeutic drugs to analyze the role of anastasis in breast cancer recurrence and metastasis after chemotherapy,and drew the following conclusions.1.Breast cancer cells can undergo anastasis after treatment with chemotherapeutic drugs.Anastasis participates in breast cancer recurrence after chemotherapy.2.Anastatic breast cancer cells exhibit enhanced proliferation and migration in vitro and increased tumor growth and metastasis in vivo.3.The enhanced proliferation and migration in anastatic breast cancer cells rely on CDH12 upregulation.The upregulated CDH12 promotes proliferation and migration through activating ERK/CREB.4.DNA methylation and repressive histone modifications are reduced in CDH12 promoter in anastatic cells.