Sam68/KHDRBS1 is critical for colon tumorigenesis by regulating genotoxic stress-induced NF-κB activation
- Bloomberg School of Public Health, Johns Hopkins University, United States
- Bloomberg School of Public Health, John Hopkins University, United States
- Johns Hopkins University School of Medicine, United States
- Johns Hopkins Medical Institutions, United States
Figures
Figure 1 with 2 supplements
Sam68 is required for the nuclear-initiated NF-κB signaling in response to DNA damage.
(A and B) Whole cell lysates from wild-type (WT) and Sam68 knockout (KO) mouse embryonic fibroblasts (MEFs) treated with indicated concentrations of CPT for 2 hr (A) or 10 μM of CPT for indicated …
Figure 1—figure supplement 1
Sam68 is critical for genotoxic stress-induced IκBα degradation.
(A) Wild-type (WT) mouse embryonic fibroblasts (MEFs), pretreated with DMSO or 10 μM of MG132 for 2 hr, were stimulated with 10 μM of CPT for indicated periods. Whole cell lysates were derived and …
Figure 1—figure supplement 2
Sam68 complexes with PARP1 and IKKγ during the cellular response to genotoxic stress.
(A) Scheme of Sam68 depletion of in CPT-stimulated wild-type mouse embryonic fibroblasts (MEFs). MEFs were stimulated with 100 μM of CPT for 0, 20, and 60 min. Under the Sam68 depletion condition, …
Figure 2 with 4 supplements
Sam68 facilitates PARP1-catalyzed PARylation in response to DNA damage.
(A) Wild-type (WT) and Sam68 knockout (KO) mouse embryonic fibroblasts (MEFs) pretreated with Olaparib (10 μM), PJ-34 (10 μM), or DMSO for 1 hr, were stimulated with 10 μM of CPT for indicated …
Figure 2—figure supplement 1
Sam68 deletion attenuates genotoxic stress-stimulated PARylation.
(A) Wild-type (WT) and Sam68 knockout (KO) mouse embryonic fibroblasts (MEFs) pretreated with PJ-34 (10 μM) or DMSO for 1 hr, were stimulated with 10 μM of CPT for indicated periods, and whole cell …
Figure 2—figure supplement 2
Down-regulation of PARG does not restore DNA damage-induced PAR production in Sam68 KO MEFs.
Wild-type (WT) and Sam68 knockout (Sam68 KO) mouse embryonic fibroblasts (MEFs) transfected with non-specific control (si-NC) or PARG-specific (si-PARG) small interference RNA, as indicated, were …
Figure 2—figure supplement 3
Sam68 functions upstream of PARP1 in cellular response to genotoxic stresses.
(A) Wild-type mouse embryonic fibroblasts (MEFs), pretreated with PJ-34 (10 μM) or DMSO for 1 hr, were γ-irradiated (IR) at 10 Gy. The chromatin fractions derived at indicated time points post …
Figure 2—figure supplement 4
Sam68-stimulated PARP1 PARylation is DNA dependent.
(A) Recombinant PARP1 protein was incubated in reaction buffer containing NAD+ and damaged DNA as indicated. The reaction mixture was separated by SDS-PAGE and subjected to immunoblot (IB) with the …
Figure 3 with 1 supplement
N-terminus of Sam68 is crucial for the Sam68-PARP1 interaction and genotoxic stress-induced NF-κB signalosome assembly.
(A) Schematic diagram of Sam68 protein (residues 1–443), full-length or indicated mutants (ΔN lacks residues 1–102, ΔC lacks 347–443, and ΔKH lacks 165–224) fused with GFP. KH, The hnRNP K homology …
Figure 3—figure supplement 1
The N-terminus-mediated Sam68-PARP1 interaction is critical for DNA damage-induced PARylation and NF-κB activation.
(A) Whole cell lysates (WCL) from HEK293T cells expressing GFP or indicated GFP-fusion proteins were immunoblotted (IB) directly or after immunoprecipitation (IP) with GFP antibody for indicated …
Figure 4 with 2 supplements
Sam68 deficiency attenuates NF-κB-mediated anti-apoptotic transcription in mouse colonic epithelial cells (CECs) under genotoxic stresses and sensitizes CECs to death.
(A) Whole cell lysates from primary Khdrbs1+/- and Khdrbs1-/- CECs treated with 10 Gy of γ-irradiation (IR) for indicated periods were immunoblotted (IB) for IκBα and Sam68, with β-actin as a …
Figure 4—figure supplement 1
Sam68 deletion attenuates genotoxic stress-induced NF-κB signaling cascade in primary mouse cells.
(A) Whole cell lysates from isolated Khdrbs1+/- (Sam68 heterozygote) and Khdrbs1-/- (Sam68 knockout) colonic epithelial cells (CECs) treated with indicated concentrations of Camptothecin (CPT) for 2 …
Figure 4—figure supplement 2
Sam68 deficiency attenuates DNA damage-triggered NF-κB-mediated expression of anti-apoptotic molecules.
(A) Wild-type (WT) and Sam68 knockout (KO) mouse embryonic fibroblasts (MEFs) were γ-irradiated (IR, 10 Gy) and total RNA was extracted at indicated time points. The mRNA profiles of Birc3, Xiap, …
Figure 5
Sam68, PAR, and NF-κB-mediated anti-apoptotic transcription are elevated in mouse and human colon cancers.
(A and E) Hematoxylin and eosin (H&E) staining and β-catenin immunohistochemistry of colon sections from tumor-loaded Apcmin716/+ mice (A) and tissue sections of colon tumor or adjacent normal colon …
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Figure 5—source data 1
Surgical colorectal cancer (CRC) and polyp metadata.
Listed are the metadata of the surgical CRC and polyp from human patients, which have been used for this research. NA, not available.
- https://doi.org/10.7554/eLife.15018.017
Figure 6 with 2 supplements
Sam68 plays a critical protective role for the survival of mouse and human colon cancers.
(A) Methylene blue (MB) staining of the colons (with cecum, proximal colon [PC], distal colon [DC], and anus indicated) derived from 3-month old Apcmin716/+; Khdrbs1+/- and Apcmin716/+; Khdrbs1-/- …
Figure 6—figure supplement 1
Sam68 knockdown and PARP inhibition attenuate PAR synthesis and PAR-dependent NF-κB transactivation in human colon cancer cell lines.
(A) T84 cells were transfected with nonspecific control (si-NC) or Sam68-specific (si-Sam68) small interference RNAs. 72 hr later, whole cell lysates were derived and immunoblotted (IB) for …
Figure 6—figure supplement 2
Sam68 knockdown sensitizes human colon cancer cells to genotoxic stress-induced apoptosis.
(A–C) HCT116 and HCT8 cells were transfected with nonspecific control (si-NC) or Sam68-specific (si-Sam68) small interference RNAs. 72 hr later, HCT116 (A) and HCT8 (B) cells were stimulated with …
Figure 7 with 1 supplement
PARP1 inhibition reduces colon tumor development in mice and sensitizes human colon cancer cells to undergo apoptosis.
(A) PARP1 inhibition in Apcmin716/+ mice in vivo. 8-week-old Apcmin716/+ mice were intraperitoneally injected with vehicle control or Olaparib (50 mg/kg) once daily for 5 days, followed by …
Figure 7—figure supplement 1
PARP1 inhibition and down-regulation of PARP1 and NF-κB triggers human cancer cells undergo apoptosis.
(A) HCT116 cells were treated with indicated concentration of PJ-34 for 72 hr. Whole cell lysates were derived and immunoblotted (IB) for indicated proteins, with β-actin as a loading control. The …
Figure 8
Schematic model representation of Sam68 functioning as an early signaling molecule in genotoxic stress-initiated NF-κB signaling pathway.
https://doi.org/10.7554/eLife.15018.023
Author response image 1
Whole cell lysates (WCL) from wild-type MEFs stimulated with 10 μM of CPT for indicated periods were immunoblotted (IB) directly or after subcellular fractionation and immunoprecipitation (IP) with Sam68 antibody for indicated proteins.
https://doi.org/10.7554/eLife.15018.024
Author response image 2
Wild-type MEFs, pretreated with DMSO or PJ-34 (10 μM) for 1h, were stimulated with 10 μM of CPT for indicated periods.
Whole cell lysates (Input) were immunoblotted (IB) directly or after immunoprecipitation (IP) with PARP1 antibody for the indicated proteins.
Author response image 3
(A) Wild-type (WT) and Sam68 knockout (KO) mouse embryonic fibroblasts (MEFs) were γ-irradiated (IR) at 4 Gy and whole cell lysates were derived at indicated time points post IR and immunoblotted …
