Ryan B. Day

CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance

Haematopoietic stem cells (HSCs) primarily reside in the bone marrow where signals generated by stromal cells regulate their self-renewal, proliferation and trafficking. Endosteal osteoblasts and perivascular stromal cells including endothelial cells, CXCL12-abundant reticular cells, leptin-receptor-positive stromal cells, and nestin–green fluorescent protein (GFP)-positive mesenchymal progenitors have all been implicated in HSC maintenance. However, it is unclear whether specific haematopoietic progenitor cell (HPC) subsets reside in distinct niches defined by the surrounding stromal cells and the regulatory molecules they produce. CXCL12 (chemokine (C–X–C motif) ligand 12) regulates both HSCs and lymphoid progenitors and is expressed by all of these stromal cell populations. Here we selectively deleted Cxcl12 from candidate niche stromal cell populations and characterized the effect on HPCs. Deletion of Cxcl12 from mineralizing osteoblasts has no effect on HSCs or lymphoid progenitors. Deletion of Cxcl12 from osterix-expressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, results in constitutive HPC mobilization and a loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from endothelial cells results in a modest loss of long-term repopulating activity. Strikingly, deletion of Cxcl12 from nestin-negative mesenchymal progenitors using Prx1–cre (Prx1 also known as Prrx1) is associated with a marked loss of HSCs, long-term repopulating activity, HSC quiescence and common lymphoid progenitors. These data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow, and that expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

Identification of Trop-2 as an oncogene and an attractive therapeutic target in colon cancers

The cell surface protein Trop-2 is highly expressed in a wide variety of epithelial cancers. In contrast, there is little or no expression of Trop-2 in adult somatic tissue. Because it is a cell surface protein that is selectively expressed in tumor cells, Trop-2 is a potential therapeutic target. However, whether Trop-2 is actively involved in tumorigenesis and whether its targeting for treatment would be effective have not been examined. Here, we show that Trop-2 expression is necessary for tumorigenesis and invasiveness of colon cancer cells, as both are inhibited when Trop-2 expression is suppressed by RNA interference. Conversely, ectopic expression of Trop-2 in colon cancer cells enhances their capacity for anchorage-independent growth and ectopic expression of Trop-2 in NIH3T3 cells is sufficient to promote both anchorage-independent growth and tumorigenesis. Importantly, we show that an antibody against the extracellular domain of Trop-2 reduces tumor cell invasiveness. Therefore, we have identified Trop-2 as an oncogene that has potential as a therapeutic target. Given the restricted expression of Trop-2 in normal tissue, anti–Trop-2 therapeutics would be predicted to have limited toxicity. [Mol Cancer Ther 2008;7(2):280–5]

Regulation of neutrophil trafficking from the bone marrow

Neutrophils are an essential component of the innate immune response and a major contributor to inflammation. Consequently, neutrophil homeostasis in the blood is highly regulated. Neutrophil number in the blood is determined by the balance between neutrophil production in the bone marrow and release from the bone marrow to blood with neutrophil clearance from the circulation. This review will focus on mechanisms regulating neutrophil release from the bone marrow. In particular, recent data demonstrating a central role for the chemokines CXCL12 and CXCL2 in regulating neutrophil egress from the bone marrow will be discussed.

Granulocyte colony-stimulating factor reprograms bone marrow stromal cells to actively suppress B lymphopoiesis in mice

The mechanisms that mediate the shift from lymphopoiesis to myelopoiesis in response to infectious stress are largely unknown. We show that treatment with granulocyte colony-stimulating factor (G-CSF), which is often induced during infection, results in marked suppression of B lymphopoiesis at multiple stages of B-cell development. Mesenchymal-lineage stromal cells in the bone marrow, including CXCL12-abundant reticular (CAR) cells and osteoblasts, constitutively support B lymphopoiesis through the production of multiple B trophic factors. G-CSF acting through a monocytic cell intermediate reprograms these stromal cells, altering their capacity to support B lymphopoiesis. G-CSF treatment is associated with an expansion of CAR cells and a shift toward osteogenic lineage commitment. It markedly suppresses the production of multiple B-cell trophic factors by CAR cells and osteoblasts, including CXCL12, kit ligand, interleukin-6, interleukin-7, and insulin-like growth factor-1. Targeting bone marrow stromal cells is one mechanism by which inflammatory cytokines such as G-CSF actively suppress lymphopoiesis.

Loss of Trop2 promotes carcinogenesis and features of epithelial to mesenchymal transition in squamous cell carcinoma.

Trop2, an oncogenic cell surface protein under investigation as a therapeutic target, is commonly overexpressed in several epithelial tumor types yet its function in tumor biology remains relatively unexplored. To investigate the role of Trop2 in epithelial carcinogenesis, we generated Trop2−/− mice, which are viable and possess a normal lifespan. Contrary to expectations, Trop2 loss fails to suppress keratinocyte transformation. Instead, ras-transformed Trop2−/− keratinocytes preferentially pass through an epithelial to mesenchymal transition (EMT) and form tumors with spindle cell histology. Furthermore, Trop2 loss renders Arf-null mice susceptible to the formation of biphasic sarcomatoid carcinomas containing both squamous and spindle cell components upon carcinogen exposure in an otherwise skin cancer–resistant strain (C57BL/6). Immortalized keratinocytes derived from Trop2−/−Arf−/− mice exhibit enhanced proliferative and migratory capacity as well as increased activation of mitogen-activated protein kinase and Src prior to transformation. The clinical relevance of these findings was supported by studying the molecular epidemiology of Trop2 in primary head and neck squamous cell carcinomas. This analysis revealed that Trop2 mRNA levels are decreased in a subset of tumors with features of EMT, and total loss of Trop2 protein expression is observed in the spindle cell component of sarcomatoid carcinomas. Therefore, while previous studies have emphasized the potential importance of Trop2 gain of function, these results uncover a role for Trop2 loss in tumorigenesis and the mesenchymal transdifferentiation observed in a subset of squamous cell carcinomas. Mol Cancer Res; 9(12); 1686–95. ©2011 AACR.

Megakaryocytes in the hematopoietic stem cell niche.

Mesenchymal stromal cells are key components of hematopoietic stem cell (HSC) niches in the bone marrow. Two studies now show that hematopoietic-derived megakaryocytes also contribute to the HSC niche, regulating HSC quiescence and function.

Restraint of the G2/M Transition by the SR/RRM Family mRNA Shuttling Binding Protein SNXAHRB1 in Aspergillus nidulans

Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated by protein–protein interactions, protein phosphorylations, and nuclear localization of CDC2/CYCLINB. We previously reported a screen, in Aspergillus nidulans, for extragenic suppressors of nimX2cdc2 that resulted in the identification of the cold-sensitive snxA1 mutation. We demonstrate here that snxA1 suppresses defects in regulators of the CDK1 mitotic induction pathway, including nimX2cdc2, nimE6cyclinB, and nimT23cdc25, but does not suppress G2-arresting nimA1/nimA5 mutations, the S-arresting nimE10cyclinB mutation, or three other G1/S phase mutations. snxA encodes the A. nidulans homolog of Saccharomyces cerevisiae Hrb1/Gbp2; nonessential shuttling messenger RNA (mRNA)-binding proteins belonging to the serine-arginine-rich (SR) and RNA recognition motif (RRM) protein family; and human heterogeneous ribonucleoprotein-M, a spliceosomal component involved in pre-mRNA processing and alternative splicing. snxAHrb1 is nonessential, its deletion phenocopies the snxA1 mutation, and its overexpression rescues snxA1 and ΔsnxA mutant phenotypes. snxA1 and a second allele isolated in this study, snxA2, are hypomorphic mutations that result from decreased transcript and protein levels, suggesting that snxA acts normally to restrain cell cycle progression. SNXAHRB1 is predominantly nuclear, but is not retained in the nucleus during the partially closed mitosis of A. nidulans. We show that the snxA1 mutation does not suppress nimX2 by altering NIMX2CDC2/NIMECYCLINB kinase activity and that snxA1 or ΔsnxA alter localization patterns of NIMECYCLINB at the restrictive temperatures for snxA1 and nimX2. Together, these findings suggest a novel and previously unreported role of an SR/RRM family protein in cell cycle regulation, specifically in control of the CDK1 mitotic induction pathway.

Abstract C167: Trop2 as a target for breast cancer treatment

Trop2 is expressed on many epithelial tumors and stem cells of the prostate and liver. Its association with aggressive cancer has led to the speculation that it might be a therapeutic target. We have previously shown a role for Trop2 in colon tumorigenesis and also found that antibodies against Trop2 can block tumor cell invasion. Consistent with this data, a monoclonal antibody against Trop2 is in pre‐clinical development against several tumor types. However, the expression pattern and role of Trop2 in breast cancer has not been examined. Here, we show heterogeneous expression of Trop2 in normal mammary gland, primary breast tumors and cancer cells lines. After purification of Trop2 expressing tumor cell populations by fluorescence activated cell sorting, we find that Trop2 hi cells are preferentially invasive and tumorigenic compared to the Trop2 lo . RNAi mediated suppression of Trop2 in breast cancer cells arrests their proliferation. Even more importantly, a monoclonal antibody against Trop2 shows anti‐tumor activity in vivo . Therefore, Trop2 represents a novel and unexplored therapeutic target in this common malignancy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C167.