Regulation of alternative splicing of Bcl-x by BC200 contributes to breast cancer pathogenesis.

BC200 is a long non-coding RNA (lncRNA) that has been implicated in the regulation of protein synthesis, yet whether dysregulation of BC200 contributes to the pathogenesis of human diseases remains elusive. In this study, we show that BC200 is upregulated in breast cancer; among breast tumor specimens there is a higher level of BC200 in estrogen receptor (ER) positive than in ER-negative tumors. Further experiments show that activation of estrogen signaling induces expression of BC200. To determine the significance of ER-regulated BC200 expression, we knockout (KO) BC200 by CRISPR/Cas9. BC200 KO suppresses tumor cell growth in vitro and in vivo by expression of the pro-apoptotic Bcl-xS isoform. Mechanistically, BC200 contains a 17-nucleotide sequence complementary to Bcl-x pre-mRNA, which may facilitate its binding to Bcl-x pre-mRNA and recruitment of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, a known splicing factor. Consequently, hnRNP A2/B1 interferes with association of Bcl-x pre-mRNA with the Bcl-xS-promoting factor Sam68, leading to a blockade of Bcl-xS expression. Together, these results suggest that BC200 plays an oncogenic role in breast cancer. Thus, BC200 may serve as a prognostic marker and possible target for attenuating deregulated cell proliferation in estrogen-dependent breast cancer.

Nuclear exclusion of p53 in a subset of tumors requires MDM2 function.

Wild type p53 accumulates in the cytoplasm in a subset of tumors such as neuroblastomas and breast carcinomas through an unknown mechanism. Exclusion of p53 from the nucleus may lead to inactivation of p53 during tumor development. We present evidence that MDM2 plays a significant role in promoting the degradation of nuclear p53 in tumor cells with a cytoplasmic p53 phenotype. Inhibition of MDM2 expression using antisense oligonucleotide, inhibition of MDM2 function by the tumor suppressor ARF or a MDM2 deletion mutant result in the accumulation of nuclear p53. p53 point mutants deficient in MDM2 binding have increased nuclear localization. Inhibition of nuclear export by leptomycin B also results in retention of nascent p53 in the nucleus, suggesting that cytoplasmic distribution of p53 results from efficient export of nuclear p53 in combination with MDM2-mediated degradation. These results suggest that MDM2 is an important determinant of p53 subcellular distribution and may contribute to p53 inactivation without overexpression.

A 60 kd MDM2 isoform is produced by caspase cleavage in non-apoptotic tumor cells.

The MDM2 oncogene product is a regulator of the p53 tumor suppressor. MDM2 is cleaved by Caspase 3 (CPP32) during apoptosis after aspartic acid-361, generating a 60 kd fragment. Here we report that human tumor cell lines often express high levels of a 60 kd MDM2 isoform (p60) in the absence of apoptosis. We demonstrate that p60 is a product of caspase cleavage of full length MDM2 after residue 361. The protease that cleaves MDM2 in non-apoptotic cells appears to be distinct from the apoptosis-specific Caspase 3, since Caspase 3 substrate poly(ADP-ribose) polymerase (PARP) is not cleaved in cells producing p60. The p60 form of MDM2 is a significant fraction of the p53-bound MDM2 protein in certain tumor cells, suggesting that it functions in the regulation of p53. p60 is also detected in breast tumors overexpressing MDM2. These observations suggest that MDM2 is regulated by caspase processing in non-apoptotic cells, and may account for the MDM2 proteins of similar mobility seen in tumors and other cell lines.

miR-509 suppresses brain metastasis of breast cancer cells by modulating RhoC and TNF-α.

The median survival time of breast cancer patients with brain metastasis is less than 6 months, and even a small metastatic lesion often causes severe neurological disabilities. Because of the location of metastatic lesions, a surgical approach is limited and most chemotherapeutic drugs are ineffective owing to the blood brain barrier (BBB). Despite this clinical importance, the molecular basis of the brain metastasis is poorly understood. In this study, we have isolated RNA from samples obtained from primary breast tumors and also from brain metastatic lesions followed by microRNA profiling analysis. Our results revealed that the miR-509 is highly expressed in the primary tumors, whereas the expression of this microRNA is significantly decreased in the brain metastatic lesions. MicroRNA target prediction and the analysis of cytokine array for the cells ectopically expressed with miR-509 demonstrated that this microRNA was capable of modulating the two genes essential for brain invasion, RhoC and TNF-α that affect the invasion of cancer cells and permeability of BBB, respectively. Importantly, high levels of TNF-α and RhoC-induced MMP9 were significantly correlated with brain metastasis-free survival of breast cancer patients. Furthermore, the results of our in vivo experiments indicate that miR-509 significantly suppressed the ability of cancer cells to metastasize to the brain. These findings suggest that miR-509 has a critical role in brain metastasis of breast cancer by modulating the RhoC-TNF-α network and that this miR-509 axis may represent a potential therapeutic target or serve as a prognostic tool for brain metastasis.

Correction of a mineralization defect by overexpression of a wild-type cDNA for COL1A1 in marrow stromal cells (MSCs) from a patient with osteogenesis imperfecta: a strategy for rescuing mutations that produce dominant-negative protein defects.

Gene therapy for dominant-negative disorders presents a more difficult challenge than gene therapy for recessive disorders, since even partial replacement of a protein for a recessive disorder can reverse symptoms. Osteogenesis imperfecta (OI) has frequently served as a model disorder for dominant-negative defects of structural proteins. The disease is caused by mutations in type I collagen (COL1A1), the major structural component of bone, skin and other connective tissues. The severity of the phenotype is largely dependent on the ratio of normal to mutant type I procollagen synthesized by cells. Recently, attempts have been made to develop strategies for cell and gene therapies using the adult stem cells from bone marrow referred to as mesenchymal stem cells or marrow stromal cells (MSCs). In this study, we used MSCs from a patient with type III OI who was heterozygous for an IVS 41A+4C mutation in COL1A1. A hybrid genomic / cDNA construct of COL1A1 was transfected into the MSCs and the transfectants were expanded over a 200-fold. Transfected MSCs showed increased expression of the wild-type mRNA and protein. In vitro assays demonstrated that the transfected cells more efficiently differentiated into mineralizing cells. The results indicated that it is possible to overexpress COL1A1 cDNA in OI MSCs and thereby to correct partially the dominant-negative protein defect.

Temperature-sensitive mutants of p53 homologs.

Two homologs of the p53 tumor suppressor, p63 and p73 have recently been discovered. These proteins have activities similar to p53 in cell culture but have distinct developmental functions in vivo. We found that temperature-sensitive mutants of certain p63 and p73 isoforms can be created by single amino acid substitutions of an alanine residue corresponding to alanine 135 of murine p53. The mutants (p63gamma-Pro167, p73alpha-Leu156 and p73beta-Ile156) can be controlled by temperature shift between 32 degrees C and 39 degrees C. They can be stably expressed in p53-null H1299 cells at 39 degrees C, become transcriptionally activated at 32 degrees C, and induce expression of p53-responsive genes MDM2 and p21WAF1. Activation of p73beta-Ile156 in H1299 cells inhibits cell division but induces significant increase in cell size (hypertrophy), whereas activation of p73alpha-Leu156 and p63gamma-Pro167 induces apoptosis. These mutants may be useful tools for gaining further insight to the functions of p53 homologs.

Activation of an MDM2-specific caspase by p53 in the absence of apoptosis.

Cells undergoing p53-mediated apoptosis activate caspase 3-like activities, resulting in the cleavage of the MDM2 oncoprotein and other apoptotic substrates such as poly(ADP-ribose) polymerase. To investigate the mechanism of p53-mediated apoptosis and to determine whether cleavage of MDM2 has a potential role in regulating p53, we examined caspase activation and cleavage of MDM2 in a cell line undergoing p53-mediated growth arrest and delayed apoptosis. We found that in H1299 cells expressing a temperature-sensitive human p53, a distinct caspase activity specific for the MDM2 cleavage site DVPD is induced by p53 prior to the onset of apoptosis and loss of viability. This is accompanied by the cleavage of MDM2 but not the apoptotic substrate poly(ADP-ribose) polymerase. The cleaved MDM2 loses the ability to promote p53 degradation and may potentially function in a dominant-negative fashion to stabilize p53. These results suggest that p53 activation may induce a positive feedback effect by cleavage of MDM2 through a unique caspase.