Journal: PLOS Biology

Article Title: The mTORC2 subunit RICTOR drives breast cancer progression by promoting ganglioside biosynthesis through transcriptional and epigenetic mechanisms

doi: 10.1371/journal.pbio.3003362

Figure Lengend Snippet: ( A ) A schematic presentation showing the ceramide-glucosylceramide rheostat connecting the sphingolipid and ganglioside metabolic pathways. ( B ) The glucosylceramide to ceramide ratio (mean ± SEM, n = 27) for luminal tumor tissues (labeled as T) and adjacent normal tissues (labeled as N) indicates that the balance is shifted towards glucosylceramides. ( C ) Heat map showing the levels of different ganglioside species in luminal tumor tissues and adjacent normal tissues. ( D – H ) Absolute quantification (mean ± SEM, n = 5) of GM3 (D), GD3 (E), GD2 (F), GM2 (G), and GM1 (H) gangliosides in luminal tumor tissues and adjacent normal tissues shows an increase in GM3 and GD3 gangliosides and a decrease in GM1 gangliosides in luminal tumor tissues. ( I ) Immunoblot confirming an increase in UGCG expression in MCF-7_UGCG OE cells. ( J ) Absolute quantification of glucosylceramides (mean ± SEM, n = 5) confirms an increase in MCF-7_UGCG OE cells over MCF-7 cells. ( K – O ) Absolute quantification (mean ± SEM, n = 5) of GM3 (K), GD3 (L), GD2 (M), GM2 (N), and GM1 (O) ganglioside species shows an increase of GM3, GD3, and GM2 gangliosides and a decrease of GM1 gangliosides in MCF-7_UGCG OE cells compared to MCF-7 cells. ( P ) Cell proliferation (mean ± SEM, n = 5) assay demonstrates an increase in the proliferation of MCF-7_UGCG OE cells over MCF-7_VECT OE cells. ( Q ) Tumor growth kinetics reveal enhanced growth of MCF-7_UGCG OE tumors compared to MCF-7_VECT OE tumors (mean ± SEM, n = 5–6). ( R ) Immunoblots show the expression of RICTOR, RAPTOR, AKT, pAKT Ser473 , SGK1, pSGK1 Ser78 , 4EBP1, p4EBP1 Thr37 , and p70S6K in MCF-7_UGCG OE cells in comparison to MCF-7_UGCG DEAD cells. ( S ) Immunoblots for RICTOR, pAKT Ser473 , and UGCG in tumor tissues from luminal cancer patients show higher expression than adjacent normal tissues. ( T ) A schematic diagram showing the questions to be answered to understand the mTORC2-mediated regulation of the sphingolipid metabolic pathway and its role in tumor progression. Data among groups were analyzed using a paired Student t test (for patient data), One-way ANOVA among multiple groups or Two-way ANOVA in time-dependent studies. P -value: * p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001. Numerical data can be found in .

Article Snippet: MCF-7, BT-474, MDA-MB-453, HCT-116, HEK-293 cells (ATCC, USA), DMEM media (Cat# D5648) Sigma, USA, MEBM media (Cat# CC-3151) Lonza, Switzerland, MEM media (Cat# AL081) HiMedia, USA, DPBS (Cat# D5652) Sigma, USA, FBS (Cat# 10270) Gibco, USA, lipid-free FBS (Cat# S148L) Biowest, USA, Penicillin-Streptomycin (Cat# 113-98-43810-74-0) HyClone, USA, Lipofectamine 2000, (Cat# 11668019) Invitrogen, USA, Lipofectamine 3000 (Cat# L3000015) Invitrogen, USA, Trypsin (Cat# TCL007) HiMedia, USA, Puromycin (Cat# P7255) Sigma, USA, G418 (Cat# A1720) Sigma, USA, Haemocytometer (Cat# Z359629) Bright-Line, USA, shRNA Control (Cat# SHC202V) Sigma, USA, shRNA RICTOR Virus Particles (Cat# SHCLNV, TRCN0000289691, TRCN0000307119, TRCN0000296313, TRCN0000307122) Sigma, USA, shRNA RICTOR Glycerol stocks (SHCLNG, TRCN0000296313, TRCN0000307122,), Sigma, USA, shRNA UGCG Glycerol stocks (SHCLNG, TRCN0000036128, TRCN0000036126, TRCN0000300623) Sigma, USA, ZFX Glycerol stocks (Cat# SHCLNG TRCN0000017308, TRCN0000017309, TRCN0000017310), Sigma, USA, shRNA ST8SIA1 (Cat# SHCLNG TRCN0000417447, TRCN0000036044, TRCN0000036046), Sigma, USA, UGCG siRNA (Cat# AM51331) Ambion, USA, ZFX siRNA (Cat# L-006572-00-0005) Dharmacon, USA, Scrambled siRNA (Cat# D-001810-10-05) Dharmacon, USA, GD3 Synthase (ST8SIA1) siRNA (Cat# EHU025731-20UG) Merck, USA, 5-Aza-2′-deoxycytidine (DAC) (Cat#A3656-10MG) Sigma, USA, KDOAM25 Hydrochloride hydrate (Cat# SML2774-5MG) Sigma, USA, MK-2206 dihydrochloride (Cat# HY-10358) MedChem Express, USA, Eliglustat (Cat# HY-14885) MedChem Express, USA, Hygromycin (Cat# PCT1503), HiMedia, USA, Puromycin (Cat# P8833), Sigma, USA.

Techniques: Labeling, Quantitative Proteomics, Western Blot, Expressing, Comparison

Journal: PLOS Biology

Article Title: The mTORC2 subunit RICTOR drives breast cancer progression by promoting ganglioside biosynthesis through transcriptional and epigenetic mechanisms

doi: 10.1371/journal.pbio.3003362

Figure Lengend Snippet: ( A ) Immunoblots confirm knockdown of RICTOR expression in MCF-7_RICTOR SH cells. ( B ) Immunoblots show changes in expression of RICTOR, RAPTOR, and their downstream effectors in MCF-7_RICTOR SH cells compared to MCF-7_SCRAM SH cells. ( C ) Cell proliferation studies show a decrease in the proliferation of MCF-7_RICTOR SH cells (mean ± SEM, n = 4) compared to MCF-7_SCRAM SH cells. ( D ) Tumor growth kinetics show significantly slower growth of MCF-7_RICTOR SH (mean ± SEM, n = 5) tumors compared to the MCF-7_SCRAM SH tumors. ( E ) Heat map representing normalized absolute quantitation of ceramides and glucosylceramides in MCF-7_RICTOR SH and MCF-7 cells. ( F ) Fold change (mean ± SEM, n = 5) in different sphingolipid species reveals an increase in ceramides and a decrease in glucosylceramides in MCF-7_RICTOR SH cells compared to MCF-7 cells. ( G , H ) qRT-PCR (mean ± SEM, n = 4) ( G ) and immunoblots and their quantification (mean ± SEM, n = 3) (H) demonstrate downregulation of UGCG without any change in GBA1 expression in MCF-7_RICTOR SH cells compared to MCF-7_SCRAM SH cells. ( I – M ) Absolute quantification (mean ± SEM, n = 3) of GM3 (I), GD3 (J), GD2 (K), GM2 (L), and GM1 (M) ganglioside species shows a decrease in GM3, GD3, GM2, and GM1 gangliosides in MCF-7_RICTOR SH cells compared to MCF-7 cells. ( N ) Immunoblot confirming UGCG overexpression in MCF-7_RICTOR SH cells. ( O ) The absolute quantification of glucosylceramides (mean ± SEM, n = 4) in MCF-7_RICTOR SH _UGCG OE cells compared to MCF-7_RICTOR SH cells confirms an increase in glucosylceramides. ( P ) Cell proliferation assay demonstrates an increase in cell proliferation (mean ± SEM, n = 4) of MCF-7_RICTOR SH cells on UGCG overexpression. ( Q ) A schematic diagram showing the role of putative factors modulating the RICTOR/pAKT-mediated UGCG expression that can lead to altered glucosylceramides, thereby controlling tumor progression. Data among groups were analyzed using an unpaired Student t test or One-way ANOVA among multiple groups or by Two-way ANOVA in time-dependent studies. p -value: * p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001. Numerical data can be found in .

Article Snippet: MCF-7, BT-474, MDA-MB-453, HCT-116, HEK-293 cells (ATCC, USA), DMEM media (Cat# D5648) Sigma, USA, MEBM media (Cat# CC-3151) Lonza, Switzerland, MEM media (Cat# AL081) HiMedia, USA, DPBS (Cat# D5652) Sigma, USA, FBS (Cat# 10270) Gibco, USA, lipid-free FBS (Cat# S148L) Biowest, USA, Penicillin-Streptomycin (Cat# 113-98-43810-74-0) HyClone, USA, Lipofectamine 2000, (Cat# 11668019) Invitrogen, USA, Lipofectamine 3000 (Cat# L3000015) Invitrogen, USA, Trypsin (Cat# TCL007) HiMedia, USA, Puromycin (Cat# P7255) Sigma, USA, G418 (Cat# A1720) Sigma, USA, Haemocytometer (Cat# Z359629) Bright-Line, USA, shRNA Control (Cat# SHC202V) Sigma, USA, shRNA RICTOR Virus Particles (Cat# SHCLNV, TRCN0000289691, TRCN0000307119, TRCN0000296313, TRCN0000307122) Sigma, USA, shRNA RICTOR Glycerol stocks (SHCLNG, TRCN0000296313, TRCN0000307122,), Sigma, USA, shRNA UGCG Glycerol stocks (SHCLNG, TRCN0000036128, TRCN0000036126, TRCN0000300623) Sigma, USA, ZFX Glycerol stocks (Cat# SHCLNG TRCN0000017308, TRCN0000017309, TRCN0000017310), Sigma, USA, shRNA ST8SIA1 (Cat# SHCLNG TRCN0000417447, TRCN0000036044, TRCN0000036046), Sigma, USA, UGCG siRNA (Cat# AM51331) Ambion, USA, ZFX siRNA (Cat# L-006572-00-0005) Dharmacon, USA, Scrambled siRNA (Cat# D-001810-10-05) Dharmacon, USA, GD3 Synthase (ST8SIA1) siRNA (Cat# EHU025731-20UG) Merck, USA, 5-Aza-2′-deoxycytidine (DAC) (Cat#A3656-10MG) Sigma, USA, KDOAM25 Hydrochloride hydrate (Cat# SML2774-5MG) Sigma, USA, MK-2206 dihydrochloride (Cat# HY-10358) MedChem Express, USA, Eliglustat (Cat# HY-14885) MedChem Express, USA, Hygromycin (Cat# PCT1503), HiMedia, USA, Puromycin (Cat# P8833), Sigma, USA.

Techniques: Western Blot, Knockdown, Expressing, Quantitation Assay, Quantitative RT-PCR, Quantitative Proteomics, Over Expression, Proliferation Assay

Journal: PLOS Biology

Article Title: The mTORC2 subunit RICTOR drives breast cancer progression by promoting ganglioside biosynthesis through transcriptional and epigenetic mechanisms

doi: 10.1371/journal.pbio.3003362

Figure Lengend Snippet: (A) A schematic diagram showing the workflow to identify RICTOR-regulated transcription factors that bind to the UGCG promoter. (B) Results from qRT-PCR (mean ± SEM, n = 3) confirm reduced expression of RICTOR-regulated ELF1 , ZFX , and CTCF transcription factors in MCF-7_RICTOR SH cells. (C) ChIP-qPCR (mean ± SEM, n = 3) results show reduced binding of ZFX to UGCG promoter in MCF-7_RICTOR SH cells. (D) EMSA shows the binding of ZFX to UGCG promoter (lanes 2 and 3) in MCF-7_ZFX OE cells, shift-ablation assay in MCF-7_ZFX OE cells (lane 4), competition assay with specific (lane 5) and unrelated oligo as a control (lane 6). “*” denotes nonspecific complexes. (E) EMSA comparing endogenous ZFX-DNA binding activity in MCF-7_SCRAM SH and MCF-7_RICTOR SH cells. (F, G) Immunoblots (F) and their quantification (mean ± SEM, n = 3) (G) confirm downregulation of ZFX in MCF-7_RICTOR SH cells. ( H , I ) Immunoblots (H) and their quantification (mean ± SEM, n = 3) (I) confirm overexpression and silencing of ZFX in MCF-7_ZFX OE and MCF-7_ZFX SL cells. (J, K) Immunoblots (J) and their quantification (mean ± SEM, n = 3) (K) show upregulation and downregulation of UGCG upon overexpression and silencing of ZFX in MCF-7_ZFX OE and MCF-7_ZFX SL cells. (L, M) Fold change (mean ± SEM, n = 5) in ceramides (L) and glucosylceramides (M) confirms a decrease in ceramides and an increase in glucosylceramides in MCF-7_ZFX OE cells. In contrast, MCF-7_ZFX SL cells show higher ceramides and reduced glucosylceramides. (N–R) Absolute quantification (mean ± SEM, n = 3-5) of GM3 (N), GD3 (O), GD2 (P), GM2 (Q), and GM1 (R) gangliosides shows an increase in GM3, GD3, and GM2 gangliosides and attenuated GM1 gangliosides on ZFX overexpression in MCF-7 cells. (S) Cell proliferation (mean ± SEM, n = 4) demonstrates increased proliferation of MCF-7_ZFX OE cells, whereas MCF-7_ZFX SL cells show reduced cell proliferation. (T) Tumor growth kinetics recorded a significantly higher growth of MCF-7_ZFX OE (mean ± SEM, n = 4-6) than MCF-7_VECT OE tumors. ( U , V ) Cell proliferation demonstrates a decrease in proliferation of MCF-7_ZFX OE cells on UGCG silencing (U) (mean ± SEM, n = 4), whereas MCF-7_ZFX SH cells show enhanced cell proliferation on UGCG overexpression (mean ± SEM, n = 3) (V). ( W ) siRNA-mediated silencing of UGCG leads to reduced tumor growth kinetics in MCF-7_ZFX OE tumors. Data among two groups were analyzed using an unpaired Student t test, among multiple groups using One-way ANOVA, and by Two-way ANOVA in time-dependent studies. p -value: * p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001. Numerical data can be found in .

Article Snippet: MCF-7, BT-474, MDA-MB-453, HCT-116, HEK-293 cells (ATCC, USA), DMEM media (Cat# D5648) Sigma, USA, MEBM media (Cat# CC-3151) Lonza, Switzerland, MEM media (Cat# AL081) HiMedia, USA, DPBS (Cat# D5652) Sigma, USA, FBS (Cat# 10270) Gibco, USA, lipid-free FBS (Cat# S148L) Biowest, USA, Penicillin-Streptomycin (Cat# 113-98-43810-74-0) HyClone, USA, Lipofectamine 2000, (Cat# 11668019) Invitrogen, USA, Lipofectamine 3000 (Cat# L3000015) Invitrogen, USA, Trypsin (Cat# TCL007) HiMedia, USA, Puromycin (Cat# P7255) Sigma, USA, G418 (Cat# A1720) Sigma, USA, Haemocytometer (Cat# Z359629) Bright-Line, USA, shRNA Control (Cat# SHC202V) Sigma, USA, shRNA RICTOR Virus Particles (Cat# SHCLNV, TRCN0000289691, TRCN0000307119, TRCN0000296313, TRCN0000307122) Sigma, USA, shRNA RICTOR Glycerol stocks (SHCLNG, TRCN0000296313, TRCN0000307122,), Sigma, USA, shRNA UGCG Glycerol stocks (SHCLNG, TRCN0000036128, TRCN0000036126, TRCN0000300623) Sigma, USA, ZFX Glycerol stocks (Cat# SHCLNG TRCN0000017308, TRCN0000017309, TRCN0000017310), Sigma, USA, shRNA ST8SIA1 (Cat# SHCLNG TRCN0000417447, TRCN0000036044, TRCN0000036046), Sigma, USA, UGCG siRNA (Cat# AM51331) Ambion, USA, ZFX siRNA (Cat# L-006572-00-0005) Dharmacon, USA, Scrambled siRNA (Cat# D-001810-10-05) Dharmacon, USA, GD3 Synthase (ST8SIA1) siRNA (Cat# EHU025731-20UG) Merck, USA, 5-Aza-2′-deoxycytidine (DAC) (Cat#A3656-10MG) Sigma, USA, KDOAM25 Hydrochloride hydrate (Cat# SML2774-5MG) Sigma, USA, MK-2206 dihydrochloride (Cat# HY-10358) MedChem Express, USA, Eliglustat (Cat# HY-14885) MedChem Express, USA, Hygromycin (Cat# PCT1503), HiMedia, USA, Puromycin (Cat# P8833), Sigma, USA.

Techniques: Quantitative RT-PCR, Expressing, ChIP-qPCR, Binding Assay, Competitive Binding Assay, Control, Activity Assay, Western Blot, Over Expression, Quantitative Proteomics

Journal: PLOS Biology

Article Title: The mTORC2 subunit RICTOR drives breast cancer progression by promoting ganglioside biosynthesis through transcriptional and epigenetic mechanisms

doi: 10.1371/journal.pbio.3003362

Figure Lengend Snippet: ( A ) Immunoblots reveal an increase in pEGFR Y1068 , pEGFR Y1173 , pAKT S473 , and pERK1/2 (Y202, Y204) in MCF-7_UGCG OE and MCF-7_ZFX OE cells compared to MCF-7 cells. ( B ) Immunoblots show attenuated EGFR activation on shRNA-mediated silencing of GD3 synthase (ST8SIA1) in MCF-7 cells. ( C ) Cell proliferation assay demonstrates a decrease in cell proliferation (mean ± SEM, n = 3) of MCF-7_ST8SIA1 SH cells compared to MCF-7 cells. ( D ) Immunoblots show enhanced EGFR activation on overexpression of ST8SIA1 in MCF-7 cells. ( E ) Cell proliferation assay demonstrates increased proliferation (mean ± SEM, n = 3) of MCF-7_ST8SIA1 OE cells compared to MCF-7 cells. ( F ) Absolute quantification (mean ± SEM, n = 3–4) of GD3 gangliosides validates the silencing and overexpression of ST8SIA1 in MCF-7 cells. ( G ) Immunoblots confirm overexpression of B3GALT4 in MCF-7 cells. ( H ) Cell proliferation assay demonstrates decreased cell proliferation (mean ± SEM, n = 4) of MCF-7_B3GALT4 OE cells compared to MCF-7 cells. ( I ) Absolute quantification (mean ± SEM, n = 5) of gangliosides validates the overexpression of B3GALT4. ( J ) Cell proliferation assay (mean ± SEM, n = 3) showing an increase in proliferation of MCF-7_RICTOR SH cells upon supplementing GD3 gangliosides and a decrease in cell proliferation upon feeding with GM1 gangliosides. ( K ) Immunoblots show attenuated EGFR activation on siRNA-mediated silencing of ST8SIA1 in MCF-7_ZFX OE cells. ( L ) Cell proliferation assay demonstrates a decrease in cell proliferation (mean ± SEM, n = 4) of MCF-7_ZFX OE cells on siRNA-mediated inhibition of ST8SIA1. ( M ) Tumor growth kinetics using xenograft studies show a decrease in growth kinetics (mean ± SEM, n = 4–6) of MCF-7_ZFX OE tumors on siRNA-mediated inhibition of ST8SIA1. Data among groups were analyzed using an unpaired Student t test, among multiple groups using One-way ANOVA, and by Two-way ANOVA in time-dependent studies. p -value: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Numerical data can be found in .

Article Snippet: MCF-7, BT-474, MDA-MB-453, HCT-116, HEK-293 cells (ATCC, USA), DMEM media (Cat# D5648) Sigma, USA, MEBM media (Cat# CC-3151) Lonza, Switzerland, MEM media (Cat# AL081) HiMedia, USA, DPBS (Cat# D5652) Sigma, USA, FBS (Cat# 10270) Gibco, USA, lipid-free FBS (Cat# S148L) Biowest, USA, Penicillin-Streptomycin (Cat# 113-98-43810-74-0) HyClone, USA, Lipofectamine 2000, (Cat# 11668019) Invitrogen, USA, Lipofectamine 3000 (Cat# L3000015) Invitrogen, USA, Trypsin (Cat# TCL007) HiMedia, USA, Puromycin (Cat# P7255) Sigma, USA, G418 (Cat# A1720) Sigma, USA, Haemocytometer (Cat# Z359629) Bright-Line, USA, shRNA Control (Cat# SHC202V) Sigma, USA, shRNA RICTOR Virus Particles (Cat# SHCLNV, TRCN0000289691, TRCN0000307119, TRCN0000296313, TRCN0000307122) Sigma, USA, shRNA RICTOR Glycerol stocks (SHCLNG, TRCN0000296313, TRCN0000307122,), Sigma, USA, shRNA UGCG Glycerol stocks (SHCLNG, TRCN0000036128, TRCN0000036126, TRCN0000300623) Sigma, USA, ZFX Glycerol stocks (Cat# SHCLNG TRCN0000017308, TRCN0000017309, TRCN0000017310), Sigma, USA, shRNA ST8SIA1 (Cat# SHCLNG TRCN0000417447, TRCN0000036044, TRCN0000036046), Sigma, USA, UGCG siRNA (Cat# AM51331) Ambion, USA, ZFX siRNA (Cat# L-006572-00-0005) Dharmacon, USA, Scrambled siRNA (Cat# D-001810-10-05) Dharmacon, USA, GD3 Synthase (ST8SIA1) siRNA (Cat# EHU025731-20UG) Merck, USA, 5-Aza-2′-deoxycytidine (DAC) (Cat#A3656-10MG) Sigma, USA, KDOAM25 Hydrochloride hydrate (Cat# SML2774-5MG) Sigma, USA, MK-2206 dihydrochloride (Cat# HY-10358) MedChem Express, USA, Eliglustat (Cat# HY-14885) MedChem Express, USA, Hygromycin (Cat# PCT1503), HiMedia, USA, Puromycin (Cat# P8833), Sigma, USA.

Techniques: Western Blot, Activation Assay, shRNA, Proliferation Assay, Over Expression, Quantitative Proteomics, Inhibition