HOXB7 Overexpression Leads Triple-Negative Breast Cancer Cells to a Less Aggressive Phenotype.

HOX genes appear to play a role in breast cancer progression in a molecular subtype-dependent way. The altered expression of HOXB7, for example, was reported to promote breast cancer progression in specific subtypes. Here we induced HOXB7 overexpression in MDA-MB-231 cells, a cellular model of the Triple-Negative breast cancer molecular subtype, and evaluated the phenotypic changes in cell viability, morphogenesis, migration, invasion, and colony formation. During the phenotypic characterization of the HOXB7-overexpressing cells, we consistently found less aggressive behavior represented by lower cell viability, inhibition of cell migration, invasion, and attachment-independent colony formation capacities added to the more compact and organized spheroid growth in 3D cultures. We then evaluated the expression of putative downstream targets and their direct binding to HOXB7 comparing ChIP-qPCR data generated from HOXB7-overexpressing cells and controls. In the manipulated cells, we found enriched biding of HOXB7 to CTNNB1, EGFR, FGF2, CDH1, DNMT3B, TGFB2, and COMMD7. Taken together, these results highlight the plasticity of the HOXB7 function in breast cancer, according to the cellular genetic background and expression levels, and provide evidence that in Triple-Negative breast cancer cells, HOXB7 overexpression has the potential to promote less aggressive phenotypes.

Dataset of HOXB7, HOXB8 and HOXB9 expression profiles in cell lines representative of the breast cancer molecular subtypes Luminal a (MCF7), Luminal b (BT474), HER2+ (SKBR3) and triple-negative (MDA231, MDA468), compared to a model of normal cells (MCF10A).

Alterations in HOXB genes expression in breast cancer have been described and related to therapy response and disease progression. However, due to breast cancer complexity and heterogeneity, added to the use of different technical approaches, the observed expression profiles are sometimes contradictory. Here, we provided the analyses of HOXB7, HOXB8 and HOXB9 expression profiles in cell lines extensively used in the literature addressing the putative role of HOXB genes in breast cancer (MCF7, BT474, SKBR3, MDA231 and MDA468) and representative of the clinical breast cancer molecular subtypes (Luminal A, Luminal B, HER2+ and Triple-negatives Claudin-low/Basal), compared to a normal breast model (MCF10A), using quantitative-PCR (qPCR). This technique allows a very sensitive quantification of gene expression and was performed using the fluorophore SYBR Green in order to obtain the expression levels relative to a reference gene, GAPDH in this case. We showed that HOXB7 is upregulated in all breast cancer cells analyzed, while HOXB8 and HOXB9 are significantly upregulated in MCF7 (Luminal A), BT474 (Luminal B) and MDA231 cells (Triple-negative Claudin-low). In addition, we found that the magnitude of the upregulation is highly subtype-specific, being the HER2+ cells the model with lowest HOXB7 upregulation, presenting very low or even null expression for HOXB8 and HOXB9, respectively. These results are analyzed in more detail in "HOX genes function in Breast Cancer development" [1] and are potentially relevant for a better understanding of the molecular heterogeneity of breast cancer, in addition to be a valuable tool assisting researchers in the choice of the most suitable cell models to perform functional assays concerning HOXB7, HOXB8 and HOXB9 genes.

HOX genes function in Breast Cancer development.

Breast cancer develops in the mammary glands during mammalian adulthood and is considered the second most common type of human carcinoma and the most incident and mortal in the female population. In contrast to other human structures, the female mammary glands continue to develop after birth, undergoing various modifications during pregnancy, lactation and involution under the regulation of hormones and transcription factors, including those encoded by the HOX clusters (A, B, C, and D). Interestingly, HOX gene deregulation is often associated to breast cancer development. Within the HOXB cluster, 8 out of the 10 genes present altered expression levels in breast cancer with an impact in its aggressiveness and resistance to hormone therapy, which highlights the importance of HOXB genes as potential therapeutic targets used to overcome the limitations of tamoxifen-resistant cancer treatments. Here, we review the current state of knowledge on the role of HOX genes in breast cancer, specially focus on HOXB, discussing the causes and consequences of HOXB gene deregulation and their relevance as prognostic factors and therapeutic targets.

Molecular ontogeny of the stomach in the catshark Scyliorhinus canicula.

The origin of extracellular digestion in metazoans was accompanied by structural and physiological alterations of the gut. These adaptations culminated in the differentiation of a novel digestive structure in jawed vertebrates, the stomach. Specific endoderm/mesenchyme signalling is required for stomach differentiation, involving the growth and transcription factors: 1) Shh and Bmp4, required for stomach outgrowth; 2) Barx1, Sfrps and Sox2, required for gastric epithelium development and 3) Cdx1 and Cdx2, involved in intestinal versus gastric identity. Thus, modulation of endoderm/mesenchyme signalling emerges as a plausible mechanism linked to the origin of the stomach. In order to gain insight into the ancient mechanisms capable of generating this structure in jawed vertebrates, we characterised the development of the gut in the catshark Scyliorhinus canicula. As chondrichthyans, these animals retained plesiomorphic features of jawed vertebrates, including a well-differentiated stomach. We identified a clear molecular regionalization of their embryonic gut, characterised by the expression of barx1 and sox2 in the prospective stomach region and expression of cdx1 and cdx2 in the prospective intestine. Furthermore, we show that gastric gland development occurs close to hatching, accompanied by the onset of gastric proton pump activity. Our findings favour a scenario in which the developmental mechanisms involved in the origin of the stomach were present in the common ancestor of chondrichthyans and osteichthyans.

The Scavenger Receptor SSc5D Physically Interacts with Bacteria through the SRCR-Containing N-Terminal Domain.

The scavenger receptor cysteine-rich (SRCR) family comprises a group of membrane-attached or secreted proteins that contain one or more modules/domains structurally similar to the membrane distal domain of type I macrophage scavenger receptor. Although no all-inclusive biological function has been ascribed to the SRCR family, some of these receptors have been shown to recognize pathogen-associated molecular patterns (PAMP) of bacteria, fungi, or other microbes. SSc5D is a recently described soluble SRCR receptor produced by monocytes/macrophages and T lymphocytes, consisting of an N-terminal portion, which contains five SRCR modules, and a large C-terminal mucin-like domain. Toward establishing a global common role for SRCR domains, we interrogated whether the set of five SRCR domains of SSc5D displayed pattern recognition receptor (PRR) properties. For that purpose, we have expressed in a mammalian expression system the N-terminal SRCR-containing moiety of SSc5D (N-SSc5D), thus excluding the mucin-like domain likely by nature to bind microorganisms, and tested the capacity of the SRCR functional groups to physically interact with bacteria. Using conventional protein-bacteria binding assays, we showed that N-SSc5D had a superior capacity to bind to Escherichia coli strains RS218 and IHE3034 compared with that of the extracellular domains of the SRCR proteins CD5 and CD6 (sCD5 and sCD6, respectively), and similar E. coli-binding properties as Spα, a proven PRR of the SRCR family. We have further designed a more sensitive, real-time, and label-free surface plasmon resonance (SPR)-based assay and examined the capacity of N-SSc5D, Spα, sCD5, and sCD6 to bind to different bacteria. We demonstrated that N-SSc5D compares with Spα in the capacity to bind to E. coli and Listeria monocytogenes, and further that it can distinguish between pathogenic E. coli RS218 and IHE3034 strains and the non-pathogenic laboratory E. coli strain BL21(DE3). Our work thus advocates the utility of SPR-based assays as sensitive tools for the rapid screening of interactions between immune-related receptors and PAMP-bearing microbes. The analysis of our results suggests that SRCR domains of different members of the family have a differential capacity to interact with bacteria, and further that the same receptor can discriminate between different bacteria strains and species.

T cell activation regulates CD6 alternative splicing by transcription dynamics and SRSF1.

The T cell-surface glycoprotein CD6 is a modulator of cellular responses and has been implicated in several autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis. During Ag presentation, CD6 is targeted to the immunological synapse in a ligand binding-dependent manner, in which CD6 domain 3 directly contacts CD166, expressed on the APC. T cell activation results in the induction of CD6Δd3, an alternatively spliced isoform that lacks the ligand-binding domain and thus no longer localizes at the immunological synapse. In this study, we investigated the molecular mechanisms regulating the expression of CD6Δd3 upon human primary T cell activation. Using chromatin immunoprecipitation, we observed an increase in RNA polymerase II occupancy along the CD6 gene and augmented CD6 transcription. We showed that activation leads to transcription-related chromatin modifications, revealed by higher CD6 acetylation levels. Modulation of chromatin conformation using a histone deacetylase inhibitor that increases transcription rate causes an increase of exon 5 skipping. We further showed that the splicing factor SRSF1 binds to a regulatory element in CD6 intron 4, activating exon 5 splicing and promoting exon 5 inclusion. Concomitant with T cell activation-induced exon 5 skipping, we observed a downregulation of SRSF1. Using RNA immunoprecipitation, we showed that in activated T cells, SRSF1 recruitment to the CD6 transcript is impaired by increased chromatin acetylation levels. We propose that upon T cell activation, SRSF1 becomes limiting, and its function in CD6 exon 5 splicing is countered by an increase in CD6 transcription, dependent on chromatin acetylation.

In vitro correction of a pseudoexon-generating deep intronic mutation in LGMD2A by antisense oligonucleotides and modified small nuclear RNAs.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is the most frequent autosomal recessive muscular dystrophy. It is caused by mutations in the calpain-3 (CAPN3) gene. The majority of the mutations described to date are located in the coding sequence of the gene. However, it is estimated that 25% of the mutations are present at exon-intron boundaries and modify the pre-mRNA splicing of the CAPN3 transcript. We have previously described the first deep intronic mutation in the CAPN3 gene: c.1782+1072G>C mutation. This mutation causes the pseudoexonization of an intronic sequence of the CAPN3 gene in the mature mRNA. In the present work, we show that the point mutation generates the inclusion of the pseudoexon in the mRNA using a minigene assay. In search of a treatment that restores normal splicing, splicing modulation was induced by RNA-based strategies, which included antisense oligonucleotides and modified small-nuclear RNAs. The best effect was observed with antisense sequences, which induced pseudoexon skipping in both HeLa cells cotransfected with mutant minigene and in fibroblasts from patients. Finally, transfection of antisense sequences and siRNA downregulation of serine/arginine-rich splicing factor 1 (SRSF1) indicate that binding of this factor to splicing enhancer sequences is involved in pseudoexon activation.

CELF proteins regulate CFTR pre-mRNA splicing: essential role of the divergent domain of ETR-3.

Cystic fibrosis is a prominent genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Among the many disease-causing alterations are pre-mRNA splicing defects that can hamper mandatory exon inclusion. CFTR exon 9 splicing depends in part on a polymorphic UG(m)U(n) sequence at the end of intron 8, which can be bound by TDP-43, leading to partial exon 9 skipping. CELF proteins, like CUG-BP1 and ETR-3, can also bind UG repeats and regulate splicing. We show here that ETR-3, but not CUG-BP1, strongly stimulates exon 9 skipping, although both proteins bind efficiently to the same RNA motif as TDP-43 and with higher affinity. We further show that the skipping of this exon may be due to the functional antagonism between U2AF65 and ETR-3 binding onto the polymorphic U or UG stretch, respectively. Importantly, we demonstrate that the divergent domain of ETR-3 is critical for CFTR exon 9 skipping, as shown by deletion and domain-swapping experiments. We propose a model whereby several RNA-binding events account for the complex regulation of CFTR exon 9 inclusion, with strikingly distinct activities of ETR-3 and CUG-BP1, related to the structure of their divergent domain.

Differential 3' splice site recognition of SMN1 and SMN2 transcripts by U2AF and U2 snRNP.

Spinal Muscular atrophy is a prevalent genetic disease caused by mutation of the SMN1 gene, which encodes the SMN protein involved in assembly of small nuclear ribonucleoprotein (snRNP) complexes. A paralog of the gene, SMN2, cannot provide adequate levels of functional SMN because exon 7 is skipped in a significant fraction of the mature transcripts. A C to T transition located at position 6 of exon 7 is critical for the difference in exon skipping between SMN1 and SMN2. Here we report that this nucleotide difference results in increased ultraviolet light-mediated crosslinking of the splicing factor U2AF(65) with the 3' splice site of SMN1 intron 6 in HeLa nuclear extract. U2 snRNP association, analyzed by native gel electrophoresis, is also more efficient on SMN1 than on SMN2, particularly under conditions of competition, suggesting more effective use of limiting factors. Two trans-acting factors implicated in SMN regulation, SF2/ASF and hnRNP A1, promote and repress, respectively, U2 snRNP recruitment to both RNAs. Interestingly, depending on the transcript and the regulatory factor, the effects on U2 binding not always correlate with changes in U2AF(65) crosslinking. Furthermore, blocking recognition of a Tra2-beta1-dependent splicing enhancer located in exon 7 inhibits U2 snRNP recruitment without affecting U2AF(65) crosslinking. Collectively, the results suggest that both U2AF binding and other steps of U2 snRNP recruitment can be control points in SMN splicing regulation.