SMAR1 suppresses the cancer stem cell population via hTERT repression in colorectal cancer cells.

One of the hallmarks of a cancer cell is the ability for indefinite proliferation leading to the immortalization of the cell. Activation of several signaling pathways leads to the immortalization of cancer cells via the reactivation of enzyme telomerase (hTERT). hTERT is active in germ cells, stem cells and also cancer cells. An earlier report from our lab suggests that SMAR1, a tumor suppressor protein, is significantly downregulated in the higher grades of colorectal cancers. Our study identifies SMAR1 as a transcriptional repressor of hTERT. We find that SMAR1 interacts with HDAC1/mSin3a co-repressor complex at the hTERT promoter and brings about HDAC1-mediated transcriptional repression of the promoter. Most solid tumors including colorectal cancer reactivate hTERT expression as it confers several advantages to the cancer cells like increased proliferation and angiogenesis. One of these non-canonical functions of hTERT is inducing the pool of cancer stem cell population. We find that in the CD133HighCD44High cancer stem cells population, SMAR1 expression is highly diminished leading to elevated hTERT expression. We also find that knockdown of SMAR1 promotes total CD133+CD44+ population and impart enhanced sphere-forming ability to the colorectal cancer cells. SMAR1 also inhibits invasion and metastasis in colorectal cancer cell lines via repression of hTERT. Our study provides evidence that downregulation of SMAR1 causes activation of hTERT leading to an increase in the cancer stem cell phenotype in colorectal cancer cells.

Tumor suppressor SMAR1 regulates PKM alternative splicing by HDAC6-mediated deacetylation of PTBP1.

BACKGROUND: Highly proliferating cancer cells exhibit the Warburg effect by regulation of PKM alternative splicing and promoting the expression of PKM2. Majority of the alternative splicing events are known to occur in the nuclear matrix where various MARBPs actively participate in the alternative splicing events. SMAR1, being a MARBP and an important tumor suppressor, is known to regulate the splicing of various cancer-associated genes. This study focuses on the regulation of PKM alternative splicing and inhibition of the Warburg effect by SMAR1. METHODS: Immunohistochemistry was performed in breast cancer patient samples to establish the correlation between SMAR1 and PKM isoform expression. Further, expression of PKM isoforms upon modulation in SMAR1 expression in breast cancer cell lines was quantified by qRT-PCR and western blot. The acetylation status of PTBP1 was estimated by immunoprecipitation along with its enrichment on PKM pre-mRNA by CLIP in SMAR1 knockdown conditions. The role of SMAR1 in tumor metabolism and tumorigenesis was explored by in vitro enzymatic assays and functional assays upon SMAR1 knockdown. Besides, in vivo tumor formation by injecting adeno-SMAR1-transduced MDA-MB-231 cells in NOD/SCID mice was performed. RESULTS: The expression profile of SMAR1 and PKM isoforms in breast cancer patients revealed that SMAR1 has an inverse correlation with PKM2 and a positive correlation with PKM1. Further quantitative PKM isoform expression upon modulation in SMAR1 expression also reflects that SMAR1 promotes the expression of PKM1 over tumorigenic isoform PKM2. SMAR1 deacetylates PTBP1 via recruitment of HDAC6 resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. SMAR1 inhibits the Warburg effect, tumorigenic potential of cancer cells, and in vivo tumor generation in a PKM2-dependent manner. CONCLUSIONS: SMAR1 regulates PKM alternative splicing by causing HDAC6-dependent deacetylation of PTBP1, resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. Additionally, SMAR1 suppresses glucose utilization and lactate production via repression of PKM2 expression. This suggests that tumor suppressor SMAR1 inhibits tumor cell metabolism and tumorigenic properties of cancer cells via regulation of PKM alternative splicing.

SMAR1 inhibits Wnt/ß-catenin signaling and prevents colorectal cancer progression.

Reduced expression of Scaffold/Matrix Attachment Region Binding Protein 1 (SMAR1) is associated with various cancers resulting in poor prognosis of the diseases. However, the precise underlying mechanism elucidating the loss of SMAR1 requires ongoing study. Here, we show that SMAR1 is highly downregulated during aberrant Wnt3a signaling due to proteasomal degradation and predicted poor prognosis of colorectal cancer. However, substitution mutation (Arginine and Lysine to Alanine) in the D-box elements of SMAR1 viz. "RCHL" and "RQRL" completely abrogated its proteasomal degradation despite Wnt3a activity. SMAR1 inhibited Wnt/ß-catenin signaling by recruiting Histone deacetylase-5 to ß-catenin promoter resulting in reduced cell migration and invasion. Consequently, reduced tumor sizes in in-vivo NOD-SCID mice were observed that strongly associated with suppression of ß-catenin. However, loss of SMAR1 led to enriched H3K9 Acetylation in the ß-catenin promoter that further increased Wnt/ß-catenin signaling activities and enhanced colorectal cancer progression drastically. Using docking and isothermal titration calorimetric studies we show that small microbial peptides viz. AT-01C and AT-01D derived from Mycobacterium tuberculosis mask the D-box elements of SMAR1. These peptides stabilized SMAR1 expression that further inhibited metastatic SW480 colorectal cancer cell migration and invasion. Drastically reduced subcutaneous tumors were observed in in-vivo NOD-SCID mice upon administration of these peptides (25 mg/kg body weight) intraperitoneally. Taken together our structural studies, in-vitro and in-vivo results strongly suggest that the D-box elements of SMAR1 represent novel druggable targets, where the microbial peptides hold promise as novel colorectal cancer therapeutics.

Photo-induced cytotoxicity and anti-metastatic activity of ruthenium(ii)-polypyridyl complexes functionalized with tyrosine or tryptophan.

The synergistic effect of oxygen, light, and photosensitizer (PS) has found applications in medicine for the treatment of cancer through photodynamic therapy (PDT). Induction of apoptosis to cancerous cells will prevent tumor metastasis that spreads cancer cells to the neighboring organs/tissues. Herein, we report the two apoptotic Ru(ii)-polypyridyl complexes that are functionalized with pendant amino acid moieties tyrosine (1) and tryptophan (2), respectively. These two water soluble complexes were found to interact strongly (K = (1.18 ± 0.28) × 105 M-1 and K = (1.57 ± 0.77) × 105 M-1) with CT-DNA. Isothermal titration calorimetry (ITC) studies revealed that these complexes bind to CT-DNA through an entropically driven process. Both the complexes showed photo-induced cytotoxicity and exhibit apoptotic activity under photo-irradiation conditions. The comet assay indicated that these complexes can damage cellular DNA, which is attributed to the significant build-up of 1O2 level even on irradiation with low intensity light (10 J cm-2, λRange 450-480 nm). This photoinduced DNA damage and apoptosis in A549 cells was induced by reactive oxygen species (ROS) and occurred through up-regulation of apoptotic marker caspase-3. Control experiments under dark conditions revealed an insignificant cytotoxicity towards these cells for two photosensitive molecules.