Fig 2: SMAR1 inhibits in vivo tumor formation. a Tumors generated in NOD/SCID mice upon injection of adeno-SMAR1-transduced MDA-MB-231 cells compared with control (n = 4). b, c Graphs representing the Tumor weight and Tumor volume. d, e Expression of PKM isoforms was checked in xenograft samples by western blot and immunohistochemistry. Error bars show mean values ± SD. Differences were considered statistically significant with *p < 0.05, **p < 0.01, and ***p < 0.001; ns non-significant difference (p > 0.05)

Fig 3: Expression of SMAR1 and PKM isoforms in breast cancer. a Expression of SMAR1, PKM1, and PKM2 in paraffinized breast tumor samples compared with normal tissue by immunohistochemistry (n = 3). b Expression profile of SMAR1, PKM1, and PKM2 in various breast cancer cell lines by western blot

Fig 4: SMAR1 binds and recruits repressor complex upstream of miR-371-373 cluster.(A) Peak visualization of ChIP-seq predicted binding of SMAR1 at miR-371-373 promoter in HCT116 p53+/+ and HCT116 p53-/- cells. (B) MAR-Wiz analysis for the miR-371-373 promoter showing presence of a potential MAR. X-axis denotes base pairs and Y-axis denotes MAR potential. (C) Chromatin immunoprecipitation (ChIP) was performed in MCF-7 cells by a-SMAR1 antibody using miR-371-373 promoter specific primers. Rabbit IgG was used as a control. Gel image shown here is the cropped version of whole gel image given in Supplementary Fig. S7. (D) ChIP followed by quantitative real time PCR (qRT PCR) was performed for detecting enrichment of SMAR1, mSin3A, HDAC1 and H3K9me3 at the miR-371-373 promoter in four cell lines, HEK293, MCF-7, MDA-MB-231 and T47D. (E) ChIP followed by quantitative real time PCR was performed for detecting enrichment of RNA polymerase II at the miR-371-373 promoter in all four cell lines. (F) The presence of histone active mark H3K9Ac was detected by ChIP-qRT PCR at the miR-371-373 promoter in all four cell lines. (G) Electrophoretic mobility shift assay (EMSA) performed for determining the presence of SMAR1 binding upstream of the miR-371-373 cluster. (H) Diagrammatic representation of pluc371 and pluc371?MAR constructs. (I) Relative luciferase activity of pluc371 and pluc371?MAR constructs upon ectopic expression and knockdown of SMAR1 as compared to control.

Fig 5: SMAR1 inhibits cancer cells in vitro and in vivo by regulating miR-371-373 cluster.(A) (i) In vitro wound healing assay in control and Adeno-SMAR1 transduced MDA-MB-231 cells showed reduced cell migration upon SMAR1 over expression. (ii) Graphical representation of the distance migrated (microns). (B) In vitro cell invasion assay performed in control MDA-MB-231 cells against different treatments of SMAR1 and miR-371-373 overexpression and/or knockdown alone and in combination. (C) In vivo imaging showing reduction in lung metastasis in mice injected with Adeno-SM transduced MDA-MB-231/Luc cells as compared to the mice injected with control MDA-MB-231/Luc cells. (D) Hematoxylin and Eosin staining of the lung sections of control mice showing alveolar collapse. Lung sections of Adeno-SM treated mice showing normal lung and alveolar architecture. (E) Quantitative real time PCR analysis showing increase in SMAR1 levels in the lung tissues of Adeno-SM treated mice. (F) Quantitative real time PCR analysis showing reduction in the miR-371-373 transcript levels in the lung tissues of Adeno-SM treated mice. (G) Kaplan-Meier relapse free survival analysis for SMAR1 in 1660 breast cancer patients. Lower expression levels of SMAR1 correlated with poor survival (p value = 0.00043). (H) Overall survival analysis for miR-371-5p in 99 breast cancer patients using MIRUMIR prediction tool. Higher expression levels corresponded to poor prognosis of patients (p value = 0.0143). (I) Disease free survival analysis for miR-373 in 99 breast carcinoma patients using MIRUMIR tool. Higher expression levels led to decreased survival of patients (p value = 0.0181). (J) Model depicting the inhibition of tumor progression and metastasis by SMAR1 mediated regulation of miR-371-373 cluster. Under normal scenario or in benign breast cancer, SMAR1 is present in physiologically normal levels in the cell thereby forming a repressor complex at the miR-371-373 promoter and repressing its expression. This allows the tumor suppressor mRNA targets of this cluster like LATS2 (Large Antigen Tumor Suppressor 2), CD44 to be translated preventing cellular transformation and maintaining homeostasis.