Research On The Activation Of Prostate Cancer Stem Cell-Like Cells By Enzalutamide-induced Senescence-associated Secretory Phenotype Factor CCL5

Prostate cancer is the most common malignant tumor of the male reproductive system.In recent years,with the growth of China's aging population and changes in living habits,the incidence of prostate cancer in Chinese men continues to rise,posing a severe threat to male health and bringing about great pressure to medical treatment.Androgen deprivation therapy?ADT?is an important treatment scheme for prostate cancer.Most patients subjected to early treatment show good reactivity,but with extended treatment time,a vast majority of patients develop resistance to androgen receptor?AR?antagonists and progress into ADT-resistant castration-resistant prostate cancer?CRPC?.At present,the highly selective AR antagonist enzalutamide?MDV3100?and other second-generation ADT drugs have been developed for CRPC and have achieved ideal clinical benefits.Patients with CRPC showed good reactivity to enzalutamide treatment,but drug resistance appeared within a short period of time.Prostate cancer develops strong resistance to ADT,which may manifest as advanced CRPC and lead to enzalutamide treatment failure.At present,studies on ADT resistance focus on signaling pathways related to AR and its mutation,which are the characteristics of prostate cancer after the occurrence of ADT resistance.However,discussion on the biological changes in prostate cancer during ADT and the possible mechanism involved in ADT resistance are still relatively limited.This study noted that during the application of ADT,prostate cancer cells showed significant senescence,a state in which cells are out of the cell cycle for a long time.Senescence is the process by which cells irreversibly stop dividing and enter a state of permanent growth arrest without undergoing cell death.Senescent cells are not in a completely static state,but show active secretion characteristics,namely senescence-associated secretory phenotype?SASP?.SASP factors include inflammatory factors,growth factors,interferons,and other molecules that are involved in pathophysiological processes such as tissue stem cell activation,tumorigenesis,inflammation,degenerative and diseases.Therefore,whether the microenvironmental changes induced by enzalutamide contribute to the occurrence of ADT resistance needs to be investigated.This study also focused on the presence of prostate cancer stem cell-like cells?PCSCs?in prostate cancer.This subgroup of cells with characteristics resembling those of stem cells,which express surface molecules such as CD44,have stronger tumorigenic capacity and are involved in prostate cancer development and drug resistance.PCSCs do not express or only express low levels of AR,leading to the lack of response to androgen and low responsiveness to ADT.According to the theory of cancer stem cells,the conversion process of prostate cancer from ADT sensitivity to ADT resistance is speculated to be accompanied by changes in the biological characteristics of PCSCs.Therefore,whether PCSCs actively respond to the biological effect of enzalutamide-induced SASP is the key to explaining the occurrence of ADT resistance.Part I:Enzalutamide-induced senescence of AR-positive LNCa P prostate cancerSenescence is a biological cell state during long-term cell cycle arrest.When treating AR-positive prostate cancer cells with enzalutamide,significant long-term cell proliferation stagnation was observed,suggesting that enzalutamide induces the senescence of prostate cancer cells.To investigate the incidence of senescence in prostate cancer cells,AR-positive LNCa P prostate cancer cells were first treated with enzalutamide and senescence-related markers were analyzed.In this study,enzalutamide significantly suppressed the proliferation of LNCa P cells and inhibited the increase in the diameter and number of 3D-cultured cells.At the same time,enzalutamide induced a significant increase in the activity of senescence-associated?-galactosidase under normal conditions and in 3D-cultured LNCa P cells.Further analysis of the expression of genes associated with the senescence signaling pathway showed that enzalutamide significantly upregulated the gene expression of P21 and P16 in LNCa P cells and that of the DNA damage marker H2AX.Expression profile sequencing showed that enzalutamide significantly inhibited biological processes and signaling pathways involved in the proliferation of LNCa P cells,such as mitosis,nuclear division,cell cycle progression,and DNA metabolism.These results showed that enzalutamide induced significant senescence in LNCa P cells.However,senescence is accompanied by obvious secretion characteristics of the SASP.As a key subgroup involved in the occurrence and development of prostate cancer,PCSCs may respond to enzalutamide-induced SASP.Further discussion on whether PCSCs undergo biological changes during enzalutamide-induced senescence will provide a direction for subsequent studies.Part II:Enzalutamide-induced activation of CD44^highPCSCs in prostate cancerThe cancer stem cell theory states that tumor growth is driven by a small number of cancer stem cells,which in some ways have similar properties as those of normal stem cells.Compared with adult stem cells,cancer stem cells show more"active"biological properties and stronger plasticity,providing a biological basis for their participation in the process of drug resistance,tumor recurrence,and metastasis.In the study of aging,adult stem cells are activated in the early stage of aging to renew cells that were lost in tissues because of cellular aging,a process driven by SASP.This section will explore the changes in the biological characteristics of stem cells in this process,such as the self-renewal of PCSCs,based on the discovery that enzalutamide induces prostate cancer cell senescence.In this study,LNCa P cells treated with enzalutamide exhibited stronger sphere formation ability.The higher expression of stemness maintenance transcription factors such as Oct4 and Sox2 indicated the increase in the stemness of the cell population.CD44-positive prostate cancer cells possess stronger self-renewal and tumorigenesis abilities and thus,CD44 is widely used in the identification of PCSCs.In this study,enzalutamide upregulated the proportion of the CD44^high subgroup of LNCa P cells and induced significantly apoptosis of CD44^lowLNCa P cells,whereas CD44^highPCSCs showed resistance to apoptosis.Therefore,whether the increase in the proportion of CD44^highPCSCs is a passive or active response remains to be further discussed.In this study,the self-renewal ability of CD44^highPCSCs was explored.Enzalutamide treatment was accompanied by enhanced CD44^highPCSC proliferation,increased expression of the cell proliferation nuclear antigen Ki67,and decreased expression of the DNA damage marker H2AX,demonstrating the enhanced self-renewal ability of CD44^highPCSCs.Meanwhile,the expression levels of the senescence-related genes P16 and P21 in CD44^highPCSCs were lower than those in CD44^lowLNCa P cells,showing a"younger"state.To explain the characteristic changes in CD44^highPCSCs comprehensively,expression profile sequencing was carried out.The results showed that enzalutamide activated cell mitosis and the Ras signaling pathway in CD44^highPCSCs,which also indicated the enhancement of self-renewal ability.This part of the study showed that enzalutamide treatment induced the activation and self-renewal enhancement of CD44^highPCSCs,whereas AR^low/-CD44^highPCSCs did not have the molecular basis for direct response to enzalutamide.The findings suggest that enzalutamide-induced SASP is an important factor in the activation of CD44^highPCSCs,which requires further investigation.Part III:Enzalutamide-induced senescence-related secretion protein CCL5 is involved in CD44^highPCSC activation and related mechanisms in prostate cancerThe secretion of SASP factors is a characteristic phenomenon of cell senescence,which causes a series of microenvironmental and pathophysiological changes.Enzalutamide can induce significant cellular senescence in prostate cancer cells,accompanied by CD44^highPCSC activation.This section will analyze the secretion characteristics of SASP factors induced by enzalutamide and explore the possible participation of SASP factors in CD44^highPCSC activation.First,the characteristics of SASP factors induced by enzalutamide in prostate cancer was analyzed using a protein chip.It was observed that the concentrations of chemokines CCL20,CCL5,CCL8,vascular endothelial growth factor,and other factors increased significantly,while those of hepatocyte growth factor,total prostate-specific antigen,and other factors decreased significantly.To screen for factors that may be involved in the activation of PCSCs,the effect of CCL5,CCL20,and CCL8 on LNCa P cell stemness maintenance-related gene expression and the percentage of CD44^highPCSCs was evaluated.It was revealed that CCL5 increased the expression of genes related to stemness maintenance.The effects of CCL5 on the biological status of CD44^highPCSCs were further investigated.CCL5 increased the proportion of CD44^highPCSCs and enhanced their proliferation ability,and these effects were partially antagonized by inhibitors of CCR5,a receptor of CCL5.The expression profile of CD44^highPCSCs induced by CCL5 showed that CCL5 activated the mitogen-activated protein kinase?MAPK?signaling pathway in CD44^highPCSC cells.Collectively,these results suggested that CCL5 plays a role in activating CD44^highPCSC cells.The characteristics of the enzalutamide-induced SASP factors in prostate cancer were analyzed using a protein microarray assay.The concentrations of CCL5 and other factors increased significantly,while those of hepatocyte growth factor,total prostate-specific antigen,and other factors decreased significantly.To screen out the factors that may be involved in activating PCSCs,the effect of SASP factors such as CCL5,CCL8,and vascular endothelial growth factor on the expression of stemness maintenance-related genes in LNCa P cells and changes in the proportion of CD44^highPCSCs was examined.CCL5 enhanced the expression of stemness maintenance genes and the proportion of CD44^highPCSCs,suggesting that CCL5 may play a role in activating PCSCs.The effect of CCL5 on the biological status of CD44^highPCSCs was further investigated.CCL5 increased the proportion of CD44^highPCSCs and enhanced their proliferation ability,and these effects were partially antagonized by inhibitors of the CCL5 receptor CCR5.At the same time,the expression profile of CD44^highPCSCs induced by CCL5 showed that CCL5 activated the MAPK signaling pathway,which is involved in the regulation of proliferation,differentiation,and other cellular behaviors.These findings suggest that CCL5 plays a role in activating CD44^highPCSCs.Both enzalutamide treatment and CCL5 induction activated the Ras and MAPK signaling pathways in CD44^highPCSCs,both of which involved the activation of downstream extracellular-signal-regulated kinase?ERK?signaling.CCR5-mediated signaling can activate ERK signaling,suggesting that ERK signaling may play a role in CCL5-activated CD44^highPCSCs.Further studies showed that enzalutamide treatment induced the expression of ERK and MEK in ERK signaling and that CCL5 induced the phosphorylation of ERK and MEK,which can be antagonized by the CCR5 inhibitor.MEK/ERK signaling is involved in the processes of self-renewal,differentiation,and mitosis of stem cells,suggesting that the CCL5/CCR5/ERK signaling pathway is critically involved in the enzalutamide-mediated activation of CD44^highPCSCs.Studies have shown that CD44^highPCSCs exhibit an active response to an ADT-induced senescence microenvironment,supporting the traditional understanding of mechanisms underlying ADT resistance.Part IV:Combination of CCR5 inhibitor maraviroc with enzalutamide synergistically inhibited tumor growth in mouse model of prostate cancerAfter receiving enzalutamide treatment,patients with prostate cancer develop ADT resistance within a short period of time,resulting in enzalutamide resistance and treatment failure.Therefore,it is of great significance to develop a method to extend the effective time window of enzalutamide treatment and improve patient prognosis.The induction of the SASP factor CCL5 by enzalutamide has the effect of activating PCSCs and may be involved in the process of enzalutamide resistance.CCL5 activates PCSCs mainly through CCR5-mediated ERK signaling and can be inhibited by an antagonist of CCR5.To this end,this section will analyze the combined therapeutic power of the CCR5 antagonist maraviroc and enzalutamide against prostate cancer.The Chou-Talalay mathematical model was applied to analyze the effect of combining the two drugs,revealing that maraviroc and enzalutamide exhibited a synergistic effect in inhibiting the proliferation of LNCa P cells.Furthermore,a subcutaneous tumor-bearing model of NOD/SCID mice with prostate cancer showed that the combined application of maraviroc further reduced the tumor-bearing quality of mice treated with enzalutamide and downregulated stem cell marker genes such as CD44 and Sox2.Meanwhile,the phosphorylation of MEK and ERK was inhibited.In conclusion,this research proposed the new viewpoint that enzalutamide induces the SASP factor CCL5 to activate CD44^highPCSCs through the CCR5 receptor and promote ADT resistance.These findings enrich the current understanding of the mechanism underlying ADT resistance and open up a new direction for the development of therapy against ADT resistance.