ACTIVATION OF TOLL-LIKE RECEPTOR 4 PROMOTES MULTIPLE MYELOMA CELL GROWTH AND SURVIVAL THROUGH UPR PATHWAY AND IN A CASPASE-DEPENDENT MANNER.
(Abstract release date: 05/19/16)
EHA Library. Kastritis E. 06/09/16; 132792; E1243
Dr. Efstathios Kastritis
Contributions
Contributions
Abstract
Abstract: E1243
Type: Eposter Presentation
Background
Toll-like receptor family (TLR) of receptors is an essential part of innate immunity. Expression and function of TLRs in Multiple Myeloma (MM) has recently become the focus of several studies and although the regulatory role of TLRs in MM plasma cells has been reported, the molecular mechanism remains unclear. During infection, human immune cells sense the presence of invading pathogens through TLRs which then activate transcriptional programs that orchestrate adaptive responses and induce the endoplasmic reticulum (ER) unfolded protein response (UPR) to accommodate essential protein translation. Studies have shown that prolonged ER stress occurs in response to microbes and specifically when cells are exposed to lipopolysaccharide (LPS), a TLR4 activator. The prolonged stress, possibly arising from a massive increase in protein synthesis, has shown to suppress CHOP, an apoptosis biomarker, in ER-stressed macrophages, while low levels of CHOP expression promote B cell survival.
Aims
The aim of our study was to investigate TLR4 signaling in myeloma cells and to explore possible implications with endoplasmic reticulum unfolded protein response as a potential mechanism of drug resistance.
Methods
mRNA expression of TLR4 and CHOP a in MM patients and cell lines was determined by real-time PCR. To deterine the effects of LPS on MM cell proliferation and apoptosis WST1 proliferation assay and Annexin-PI staining was used respectively. Protein expression of CHOP, ATF4, peIF2a, NFkB and caspases was assesed by immunoblotting analysis.Bottom of Form
Results
We found that TLR4 mRNA is expressed at increased levels (2-10 fold) both in human myeloma cell lines (HMCL) and primary cells. To investigate whether TLR4 signaling may suppress CHOP expression during sustained UPR response JJN3 and U266 cells, were pre-treated with LPS (5 ug/mL) and then subjected to ER stress conditions with a typical ER stressor, tunicamycin (TM). We found that ER stress-induced CHOP expression was suppressed by prior engagement of TLR4, by LPS pre-treatment. To test the relevance of these results, pre-treated LPS and TM samples were also subjected to Annexin-PI staining to determine the amount of apoptosis. As expected, pre-treated LPS HMCLs exposed to TM had almost 30% lower Annexin-FITC stained cells compared to the TM-stressed cells only. Thus, downregulation of CHOP by TLR4 ligands may confer resistance to apoptotic stimuli and promote the growth and survival of MM cells. To determine the potential therapeutic impact of TLR4 signaling in myeloma, we evaluated the activity of bortezomib in LPS pre-treated above cell lines .LPS pre-treatment partially abrogated the efficacy of bortezomib in these cell lines by decreasing its anti-proliferative effects compared to the non-LPS-pretreated cell lines as tested by the WST1 assay. In addition extract of cells were immunoblotted for CHOP protein expression. Pre-treatment of MM cells with low dose LPS selectively suppressed CHOP in bortezomib treated cells compared to the non-LPS treated cells. CHOP suppression was also accompanied with suppression of PERK, ATF4 and peiF2a, protein expression. In order to determine whether the escape from the apoptotic pathway which was achieved by LPS was preceded in caspase-dependent manner, the protein expression of caspase-8 and caspase-9 was explored upon treatment with LPS. Indeed, LPS stimulation was able to partially inhibit caspase-8 and caspase-9 expression in bortezomib-induced JJN3 cells. Furthemore pretreatment of LPS was able to completely abort NFκB inhibition induced by bortezomib.Then, we examined the impact bortezomib therapy on TLR4 and CHOP mRNA expression in primary tumors cells, collected before and at day 7 after bortezomib-based therapy from 6 myeloma patients. In 5 out of 6 cases TLR4 expression was significantly up-regulated and was accompanied with a coupled down-regulation of CHOP mRNA expression.
Conclusion
In conclusion, our data suggest that the TLR4 signaling pathway might provide a translational control pathway which disables cells to undergo the programmed cell death by apoptosis via CHOP branch. Further exploration of this pathway is needed to establish its role as a potential mechanism of drug resistance of MM cells which may direct future development of novel therapeutic targets.
Session topic: E-poster
Type: Eposter Presentation
Background
Toll-like receptor family (TLR) of receptors is an essential part of innate immunity. Expression and function of TLRs in Multiple Myeloma (MM) has recently become the focus of several studies and although the regulatory role of TLRs in MM plasma cells has been reported, the molecular mechanism remains unclear. During infection, human immune cells sense the presence of invading pathogens through TLRs which then activate transcriptional programs that orchestrate adaptive responses and induce the endoplasmic reticulum (ER) unfolded protein response (UPR) to accommodate essential protein translation. Studies have shown that prolonged ER stress occurs in response to microbes and specifically when cells are exposed to lipopolysaccharide (LPS), a TLR4 activator. The prolonged stress, possibly arising from a massive increase in protein synthesis, has shown to suppress CHOP, an apoptosis biomarker, in ER-stressed macrophages, while low levels of CHOP expression promote B cell survival.
Aims
The aim of our study was to investigate TLR4 signaling in myeloma cells and to explore possible implications with endoplasmic reticulum unfolded protein response as a potential mechanism of drug resistance.
Methods
mRNA expression of TLR4 and CHOP a in MM patients and cell lines was determined by real-time PCR. To deterine the effects of LPS on MM cell proliferation and apoptosis WST1 proliferation assay and Annexin-PI staining was used respectively. Protein expression of CHOP, ATF4, peIF2a, NFkB and caspases was assesed by immunoblotting analysis.Bottom of Form
Results
We found that TLR4 mRNA is expressed at increased levels (2-10 fold) both in human myeloma cell lines (HMCL) and primary cells. To investigate whether TLR4 signaling may suppress CHOP expression during sustained UPR response JJN3 and U266 cells, were pre-treated with LPS (5 ug/mL) and then subjected to ER stress conditions with a typical ER stressor, tunicamycin (TM). We found that ER stress-induced CHOP expression was suppressed by prior engagement of TLR4, by LPS pre-treatment. To test the relevance of these results, pre-treated LPS and TM samples were also subjected to Annexin-PI staining to determine the amount of apoptosis. As expected, pre-treated LPS HMCLs exposed to TM had almost 30% lower Annexin-FITC stained cells compared to the TM-stressed cells only. Thus, downregulation of CHOP by TLR4 ligands may confer resistance to apoptotic stimuli and promote the growth and survival of MM cells. To determine the potential therapeutic impact of TLR4 signaling in myeloma, we evaluated the activity of bortezomib in LPS pre-treated above cell lines .LPS pre-treatment partially abrogated the efficacy of bortezomib in these cell lines by decreasing its anti-proliferative effects compared to the non-LPS-pretreated cell lines as tested by the WST1 assay. In addition extract of cells were immunoblotted for CHOP protein expression. Pre-treatment of MM cells with low dose LPS selectively suppressed CHOP in bortezomib treated cells compared to the non-LPS treated cells. CHOP suppression was also accompanied with suppression of PERK, ATF4 and peiF2a, protein expression. In order to determine whether the escape from the apoptotic pathway which was achieved by LPS was preceded in caspase-dependent manner, the protein expression of caspase-8 and caspase-9 was explored upon treatment with LPS. Indeed, LPS stimulation was able to partially inhibit caspase-8 and caspase-9 expression in bortezomib-induced JJN3 cells. Furthemore pretreatment of LPS was able to completely abort NFκB inhibition induced by bortezomib.Then, we examined the impact bortezomib therapy on TLR4 and CHOP mRNA expression in primary tumors cells, collected before and at day 7 after bortezomib-based therapy from 6 myeloma patients. In 5 out of 6 cases TLR4 expression was significantly up-regulated and was accompanied with a coupled down-regulation of CHOP mRNA expression.
Conclusion
In conclusion, our data suggest that the TLR4 signaling pathway might provide a translational control pathway which disables cells to undergo the programmed cell death by apoptosis via CHOP branch. Further exploration of this pathway is needed to establish its role as a potential mechanism of drug resistance of MM cells which may direct future development of novel therapeutic targets.
Session topic: E-poster
Abstract: E1243
Type: Eposter Presentation
Background
Toll-like receptor family (TLR) of receptors is an essential part of innate immunity. Expression and function of TLRs in Multiple Myeloma (MM) has recently become the focus of several studies and although the regulatory role of TLRs in MM plasma cells has been reported, the molecular mechanism remains unclear. During infection, human immune cells sense the presence of invading pathogens through TLRs which then activate transcriptional programs that orchestrate adaptive responses and induce the endoplasmic reticulum (ER) unfolded protein response (UPR) to accommodate essential protein translation. Studies have shown that prolonged ER stress occurs in response to microbes and specifically when cells are exposed to lipopolysaccharide (LPS), a TLR4 activator. The prolonged stress, possibly arising from a massive increase in protein synthesis, has shown to suppress CHOP, an apoptosis biomarker, in ER-stressed macrophages, while low levels of CHOP expression promote B cell survival.
Aims
The aim of our study was to investigate TLR4 signaling in myeloma cells and to explore possible implications with endoplasmic reticulum unfolded protein response as a potential mechanism of drug resistance.
Methods
mRNA expression of TLR4 and CHOP a in MM patients and cell lines was determined by real-time PCR. To deterine the effects of LPS on MM cell proliferation and apoptosis WST1 proliferation assay and Annexin-PI staining was used respectively. Protein expression of CHOP, ATF4, peIF2a, NFkB and caspases was assesed by immunoblotting analysis.Bottom of Form
Results
We found that TLR4 mRNA is expressed at increased levels (2-10 fold) both in human myeloma cell lines (HMCL) and primary cells. To investigate whether TLR4 signaling may suppress CHOP expression during sustained UPR response JJN3 and U266 cells, were pre-treated with LPS (5 ug/mL) and then subjected to ER stress conditions with a typical ER stressor, tunicamycin (TM). We found that ER stress-induced CHOP expression was suppressed by prior engagement of TLR4, by LPS pre-treatment. To test the relevance of these results, pre-treated LPS and TM samples were also subjected to Annexin-PI staining to determine the amount of apoptosis. As expected, pre-treated LPS HMCLs exposed to TM had almost 30% lower Annexin-FITC stained cells compared to the TM-stressed cells only. Thus, downregulation of CHOP by TLR4 ligands may confer resistance to apoptotic stimuli and promote the growth and survival of MM cells. To determine the potential therapeutic impact of TLR4 signaling in myeloma, we evaluated the activity of bortezomib in LPS pre-treated above cell lines .LPS pre-treatment partially abrogated the efficacy of bortezomib in these cell lines by decreasing its anti-proliferative effects compared to the non-LPS-pretreated cell lines as tested by the WST1 assay. In addition extract of cells were immunoblotted for CHOP protein expression. Pre-treatment of MM cells with low dose LPS selectively suppressed CHOP in bortezomib treated cells compared to the non-LPS treated cells. CHOP suppression was also accompanied with suppression of PERK, ATF4 and peiF2a, protein expression. In order to determine whether the escape from the apoptotic pathway which was achieved by LPS was preceded in caspase-dependent manner, the protein expression of caspase-8 and caspase-9 was explored upon treatment with LPS. Indeed, LPS stimulation was able to partially inhibit caspase-8 and caspase-9 expression in bortezomib-induced JJN3 cells. Furthemore pretreatment of LPS was able to completely abort NFκB inhibition induced by bortezomib.Then, we examined the impact bortezomib therapy on TLR4 and CHOP mRNA expression in primary tumors cells, collected before and at day 7 after bortezomib-based therapy from 6 myeloma patients. In 5 out of 6 cases TLR4 expression was significantly up-regulated and was accompanied with a coupled down-regulation of CHOP mRNA expression.
Conclusion
In conclusion, our data suggest that the TLR4 signaling pathway might provide a translational control pathway which disables cells to undergo the programmed cell death by apoptosis via CHOP branch. Further exploration of this pathway is needed to establish its role as a potential mechanism of drug resistance of MM cells which may direct future development of novel therapeutic targets.
Session topic: E-poster
Type: Eposter Presentation
Background
Toll-like receptor family (TLR) of receptors is an essential part of innate immunity. Expression and function of TLRs in Multiple Myeloma (MM) has recently become the focus of several studies and although the regulatory role of TLRs in MM plasma cells has been reported, the molecular mechanism remains unclear. During infection, human immune cells sense the presence of invading pathogens through TLRs which then activate transcriptional programs that orchestrate adaptive responses and induce the endoplasmic reticulum (ER) unfolded protein response (UPR) to accommodate essential protein translation. Studies have shown that prolonged ER stress occurs in response to microbes and specifically when cells are exposed to lipopolysaccharide (LPS), a TLR4 activator. The prolonged stress, possibly arising from a massive increase in protein synthesis, has shown to suppress CHOP, an apoptosis biomarker, in ER-stressed macrophages, while low levels of CHOP expression promote B cell survival.
Aims
The aim of our study was to investigate TLR4 signaling in myeloma cells and to explore possible implications with endoplasmic reticulum unfolded protein response as a potential mechanism of drug resistance.
Methods
mRNA expression of TLR4 and CHOP a in MM patients and cell lines was determined by real-time PCR. To deterine the effects of LPS on MM cell proliferation and apoptosis WST1 proliferation assay and Annexin-PI staining was used respectively. Protein expression of CHOP, ATF4, peIF2a, NFkB and caspases was assesed by immunoblotting analysis.Bottom of Form
Results
We found that TLR4 mRNA is expressed at increased levels (2-10 fold) both in human myeloma cell lines (HMCL) and primary cells. To investigate whether TLR4 signaling may suppress CHOP expression during sustained UPR response JJN3 and U266 cells, were pre-treated with LPS (5 ug/mL) and then subjected to ER stress conditions with a typical ER stressor, tunicamycin (TM). We found that ER stress-induced CHOP expression was suppressed by prior engagement of TLR4, by LPS pre-treatment. To test the relevance of these results, pre-treated LPS and TM samples were also subjected to Annexin-PI staining to determine the amount of apoptosis. As expected, pre-treated LPS HMCLs exposed to TM had almost 30% lower Annexin-FITC stained cells compared to the TM-stressed cells only. Thus, downregulation of CHOP by TLR4 ligands may confer resistance to apoptotic stimuli and promote the growth and survival of MM cells. To determine the potential therapeutic impact of TLR4 signaling in myeloma, we evaluated the activity of bortezomib in LPS pre-treated above cell lines .LPS pre-treatment partially abrogated the efficacy of bortezomib in these cell lines by decreasing its anti-proliferative effects compared to the non-LPS-pretreated cell lines as tested by the WST1 assay. In addition extract of cells were immunoblotted for CHOP protein expression. Pre-treatment of MM cells with low dose LPS selectively suppressed CHOP in bortezomib treated cells compared to the non-LPS treated cells. CHOP suppression was also accompanied with suppression of PERK, ATF4 and peiF2a, protein expression. In order to determine whether the escape from the apoptotic pathway which was achieved by LPS was preceded in caspase-dependent manner, the protein expression of caspase-8 and caspase-9 was explored upon treatment with LPS. Indeed, LPS stimulation was able to partially inhibit caspase-8 and caspase-9 expression in bortezomib-induced JJN3 cells. Furthemore pretreatment of LPS was able to completely abort NFκB inhibition induced by bortezomib.Then, we examined the impact bortezomib therapy on TLR4 and CHOP mRNA expression in primary tumors cells, collected before and at day 7 after bortezomib-based therapy from 6 myeloma patients. In 5 out of 6 cases TLR4 expression was significantly up-regulated and was accompanied with a coupled down-regulation of CHOP mRNA expression.
Conclusion
In conclusion, our data suggest that the TLR4 signaling pathway might provide a translational control pathway which disables cells to undergo the programmed cell death by apoptosis via CHOP branch. Further exploration of this pathway is needed to establish its role as a potential mechanism of drug resistance of MM cells which may direct future development of novel therapeutic targets.
Session topic: E-poster
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