Rab31 upregulates MUC1-C expression.

<p>A. MCF-7 (left) and ZR-75-1 (right) cells were stably transfected to express a control siRNA (CsiRNA) or a Rab31 siRNA. Lysates from the transfectants were immunoblotted with the indicated antibodies. B. MCF-7/Rab31siRNA (left) and ZR-75-1/Rab31siRNA (right) cells were treated with the indicated concentrations of chloroquine (CQ) for 24 h. Lysates from the treated cells were immunoblotted with the indicated antibodies. C. RNA from MCF-7, ZR-75-1 and MCF-10A cells was analyzed for Rab31 mRNA levels by qRT-PCR. The results (mean<u>+</u>SD of three determinations) are expressed as relative Rab31 mRNA levels compared to that obtained for MCF-7 cells. D. Lysates from MCF-7, ZR-75-1 and MCF-10A cells were immunoblotted with the indicated antibodies. E. MCF-10A cells were stably transfected to express an empty vector, Rab31 or Rab31(S20N). Lysates from the transfected cells were immunoblotted with the indicated antibodies.</p

MUC1 induces Rab31 expression.

<p>A and B. RNA from the indicated MCF-7 (A) and ZR-75-1 (B) cells was analyzed by RT-PCR using primers designed to detect the indicated transcripts (left). RNA was also analyzed for Rab31 mRNA levels by qRT-PCR (right). The results (mean±SD of three determinations) are expressed as relative Rab31 mRNA levels compared to that obtained in the vector cells. C and D. Lysates from the indicated MCF-7 (C) and ZR-75-1 (D) cells were immunoblotted with the indicated antibodies.</p

Activation of the <i>Rab31</i> promoter by MUC1 and ERα.

<p>A. Schematic representation of the <i>Rab31</i> promoter with localization of putative estrogen response elements (EREs). B and C. The indicated MCF-7 (B) and ZR-75-1 (C) cells were transfected with pRab31-Luc and <i>Renilla</i> plasmids for 48 h and then assayed for luciferase activity. The results are expressed as the relative luciferase activity (mean±SD of three determinations) compared to that obtained in the vector cells. D. MCF-7 cells were transiently transfected with control and ERα siRNA pools. Lysates were immunoblotted with the indicated antibodies (left). The cells were also transfected with pRab31-Luc and <i>Renilla</i> plasmids for 48 h. The results are expressed as the relative luciferase activity (mean±SD of three determinations) compared to that obtained in the CsiRNA-transfected cells (right). E. ZR-75-1 cells were transfected with pRab31-Luc and <i>Renilla</i> plasmids for 24 h. The cells were then cultured in phenol red-free medium with 2% charcoal dextran-treated serum for 72 h, treated with E2 for 24 h and assayed for luciferase activity. The results are expressed as the relative luciferase activity (mean±SD of three determinations) compared to that obtained in the untreated control (CTL) cells.</p

Expression of Rab31 in human breast cancers.

<p>A. Analysis of Rab31 mRNA levels in the GSE5764 dataset from 23 normal breast tissues and 10 breast tumors. The results are expressed as the relative Rab31 mRNA levels based on the normalization values in the dataset. B. Analysis of MUC1 mRNA (left) and Rab31 mRNA (right) levels in the GSE5460 dataset from 76 ER+ and 53 ER− breast tumors. Normalized microarray data were separated into ER+ and ER- groups. C and D. Analysis of MUC1 mRNA (left), Rab31 mRNA (middle) and co-expression levels (right) in the Loi dataset (C, 262 ER+ and 45 ER− breast tumors) and van de Vijver dataset (D, 226 ER+ and 69 ER− breast tumors). E. Analysis of Rab31 and MUC1 mRNA co-expression levels in 147 ER+ breast tumors (left). Percentage overall survival for patients with Rab31-positive versus Rab31-negative breast tumors (right; Chanrion dataset).</p

Rab31 promotes MCF-10A mammosphere formation by a MUC1-C-dependent mechanism.

<p>A. The indicated MCF-10A cells were seeded in soft agar. After culturing for 3 weeks, photomicrographs were obtained (left) and the numbers of colonies (mean±SD of three replicates) were determined by counting clusters of >20 cells. B. The indicated MCF-10A cells were seeded under conditions for the growth of mammospheres. After culturing for 3 weeks, photomicrographs were obtained (left) and the numbers of mammospheres (mean±SD of three replicates) were determined by counting spheres of >20 cells (right). C. Lysates from MCF-10A/Rab31 cells grown as an adherent monolayer and as mammospheres were immunoblotted with the indicated antibodies (left). MCF-10A/Rab31 cells were transfected with control and MUC1 siRNAs. Lysates were immunoblotted with the indicated antibodies (right). D. MCF-10A/Rab31 cells transfected with the indicated siRNAs were cultured for the growth of mammospheres. After 3 weeks, photomicrographs were obtained (left) and the numbers of mammospheres (mean±SD of three replicates) were determined by counting spheres of >20 cells (right). E. MCF-10A/Rab31 cells were cultured for the growth of mammospheres in the absence (Control) and presence of 5 µM GO-203 or CP-2. After 3 weeks, photomicrographs were obtained (left) and the numbers of mammospheres (mean±SD of three replicates) were determined by counting spheres of >20 cells (right).</p

MUC1-C associates with ERα on the <i>Rab31</i> promoter.

<p>A and B. Lysates from MCF-7 (A) and ZR-75-1 (B) cells left untreated or stimulated with E2 for 24 h were immunoprecipitated with anti-ERα or a control IgG. The precipitates were immunoblotted with anti-MUC1-C or anti-ERα. C. Schema of the <i>Rab31</i> promoter highlighting the positions of the control region (CR) and the proximal region encompassing estrogen response elements (EREs). D and E. Soluble chromatin from MCF-7 (D) and ZR-75-1 (E) cells left untreated or stimulated with E2 for 24 h was precipitated with a control IgG or anti-ERα. The precipitates were analyzed for <i>Rab31</i> promoter ERE or CR sequences (left). The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared to that obtained with the IgG control. In re-ChIP experiments, the anti-ERα precipitates were released, reimmunoprecipitated with anti-MUC1-C, and then analyzed for <i>Rab31</i> promoter sequences (right). The results (mean±SD of three determinations) are expressed as the relative fold enrichment compared to that obtained with the unstimulated control.</p

Porous Particle-Reinforced Bioactive Gelatin Scaffold for Large Segmental Bone Defect Repairing

Large segmental bone defect repairing remains a big challenge in clinics, and synthetic bone grafts suitable for this purpose are still highly demanded. In this article, hydrophilic composite scaffolds (bioactive hollow particle (BHP)–gel scaffold) composed of bioactive hollow nanoparticles and cross-linked gelatin have been developed. The bioactive nanoparticles have a porous structure as well as high specific surface area; thus, they interact strongly with gelatin to overcome the swelling problem that a hydrophilic polymer scaffold will usually face. With this combination, these BHP–gel scaffolds showed porous structure and mechanical properties similar to those of the cancellous bone. They also showed excellent bioactivity and cell growth promotion performance in vitro. The best of them, namely, 10BHP-gel scaffold, was evaluated in vivo on a rat femur model, where it was found that the 5 mm segmental bone defect almost healed with new bone tissue formed in 12 weeks and the scaffold itself degraded at the same time. Thus, 10BHP-gel scaffold may become a potential bone graft for large segmental bone defect healing in the future

Discovery of Orally Active Inhibitors of Brahma Homolog (BRM)/SMARCA2 ATPase Activity for the Treatment of Brahma Related Gene 1 (BRG1)/SMARCA4-Mutant Cancers

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2), also known as Brahma homologue (BRM), is a Snf2-family DNA-dependent ATPase. BRM and its close homologue Brahma-related gene 1 (BRG1), also known as SMARCA4, are mutually exclusive ATPases of the large ATP-dependent SWI/SNF chromatin-remodeling complexes involved in transcriptional regulation of gene expression. No small molecules have been reported that modulate SWI/SNF chromatin-remodeling activity via inhibition of its ATPase activity, an important goal given the well-established dependence of BRG1-deficient cancers on BRM. Here, we describe allosteric dual BRM and BRG1 inhibitors that downregulate BRM-dependent gene expression and show antiproliferative activity in a BRG1-mutant-lung-tumor xenograft model upon oral administration. These compounds represent useful tools for understanding the functions of BRM in BRG1-loss-of-function settings and should enable probing the role of SWI/SNF functions more broadly in different cancer contexts and those of other diseases