Mira C.P. Liu

Hemopoietic Stem Cells with Higher Hemopoietic Potential Reside at the Bone Marrow Endosteum

It is now evident that hemopoietic stem cells (HSC) are located in close proximity to bone lining cells within the endosteum. Accordingly, it is unlikely that the traditional method for harvesting bone marrow (BM) from mice by simply flushing long bones would result in optimal recovery of HSC. With this in mind, we have developed improved methodologies based on sequential grinding and enzymatic digestion of murine bone tissue to harvest higher numbers of BM cells and HSC from the endosteal and central marrow regions. This methodology resulted in up to a sixfold greater recovery of primitive hemopoietic cells (lineage−Sca+Kit+ [LSK] cells) and HSC as shown by transplant studies. HSC from different anatomical regions of the marrow exhibited important functional differences. Compared with their central marrow counterparts, HSC isolated from the endosteal region (a) had 1.8‐fold greater proliferative potential, (b) exhibited almost twofold greater ability to home to the BM following tail vein injection and to lodge in the endosteal region, and (c) demonstrated significantly greater long‐term hemopoietic reconstitution potential as shown using limiting dilution competitive transplant assays.

Inhibition of Siah ubiquitin ligase function

Tumor hypoxia induces the upregulation of hypoxia-inducible factor 1α (Hif-1α), which in turn induces the expression of genes including VEGF to recruit new blood vessel outgrowth, enabling tumor growth and metastasis. Interference with the Hif-1 pathway and neoangiogenesis is an attractive antitumor target. The hydroxylation of Hif-1α by prolyl-hydroxylase (PHD) proteins during normoxia serves as a recognition motif for its proteasomal degradation. However, under hypoxic conditions, hydroxylation is inhibited and furthermore, PHD proteins are themselves polyubiquitylated and degraded by Siah ubiquitin ligases. Our data demonstrate for the first time that inhibition of the interaction between Siah and PHD proteins using a fragment derived from a Drosophila protein (phyllopod) interferes with the PHD degradation. Furthermore, cells stably expressing the phyllopod fragment display reduced upregulation of Hif-1α protein levels and Hif-1-mediated gene expression under hypoxia. In a syngeneic mouse model of breast cancer, the phyllopod fragment reduced tumor growth and neoangiogenesis and prolonged survival of the mice. In addition, levels of Hif-1α and its target Glut-1 are reduced in tumors expressing the phyllopod fragment. These data show, in a proof-of-principle study, that Siah protein, the most upstream component of the hypoxia pathway yet identified, is a viable drug target for antitumor therapies.

Vascular Normalization by Loss of Siah2 Results in Increased Chemotherapeutic Efficacy

Tumor hypoxia is associated with resistance to antiangiogenic therapy and poor prognosis. The Siah E3 ubiquitin ligases regulate the hypoxic response pathway by modulating the turnover of the master proangiogenic transcription factor hypoxia-inducible factor-1α (Hif-1α). In this study, we show that genetic deficiency in the Siah family member Siah2 results in vascular normalization and delayed tumor growth in an established transgenic model of aggressive breast cancer. Tumors arising in a Siah2(-/-) genetic background showed increased perfusion and pericyte-associated vasculature, similar to that occurring with antiangiogenic therapy. In support of the role of Siah2 in regulating levels of Hif-1α, expression of angiogenic factors was decreased in Siah2(-/-) tumors. Blood vessel normalization in Siah2(-/-) tumors resulted in an increased response to chemotherapy and prolonged survival. Together, our findings offer a preclinical proof of concept that targeting Siah2 is sufficient to attenuate Hif-1α-mediated angiogenesis and hypoxia signaling, thereby improving responses to chemotherapy.

The expression of the ubiquitin ligase SIAH2 (seven in absentia homolog 2) is mediated through gene copy number in breast cancer and is associated with a basal-like phenotype and p53 expression.

IntroductionThe seven in absentia homolog 2 (SIAH2) protein plays a significant role in the hypoxic response by regulating the abundance of hypoxia-inducible factor-α; however, its role in breast carcinoma is unclear. We investigated the frequency and expression pattern of SIAH2 in two independent cohorts of sporadic breast cancers.MethodsImmunohistochemical evaluation of SIAH2protein expression was conducted in normal breast tissues and in tissue microarrays comprising ductal carcinoma in situ (DCIS) and a cohort of invasive breast carcinomas. Correlation analysis was performed between SIAH2 and clinicopathological variables and intrinsic breast cancer subgroups and validated in a cohort of 293 invasive ductal carcinomas. Promoter methylation, gene copy number and mRNA expression of SIAH2 were determined in a panel of basal-like tumors and cell lines.ResultsThere was a significant increase in nuclear SIAH2 expression from normal breast tissues through to DCIS and progression to invasive cancers. A significant inverse correlation was apparent between SIAH2 and estrogen receptor and progesterone receptor and a positive association with tumor grade, HER2, p53 and an intrinsic basal-like subtype. Logistic regression analysis confirmed the significant positive association between SIAH2 expression and the basal-like phenotype. No SIAH2 promoter methylation was identified, yet there was a significant correlation between SIAH2 mRNA and gene copy number. SIAH2-positive tumors were associated with a shorter relapse-free survival in univariate but not multivariate analysis.ConclusionsSIAH2 expression is upregulated in basal-like breast cancers via copy number changes and/or transcriptional activation by p53 and is likely to be partly responsible for the enhanced hypoxic drive through abrogation of the prolyl hydroxylases.

Developmental Fate Determination and Marker Discovery in Hematopoietic Stem Cell Biology Using Proteomic Fingerprinting

In hematopoiesis, co-expression of Sca-1 and c-Kit defines cells (LS+K) with long term reconstituting potential. In contrast, poorly characterized LS−K cells fail to reconstitute lethally irradiated recipients. Relative quantification mass spectrometry and transcriptional profiling were used to characterize LS+K and LS−K cells. This approach yielded data on >1200 proteins. Only 32% of protein changes correlated to mRNA modulation demonstrating post-translational protein regulation in early hematopoietic development. LS+K cells had lower expression of protein synthesis proteins but did express proteins associated with mature cell function. Major increases in erythroid development proteins were observed in LS−K cells; based on this assessment of erythroid potential we showed them to be principally erythroid progenitors, demonstrating effective use of discovery proteomics for definition of primitive cells.

The Antioxidant N-Acetylcysteine Prevents HIF-1 Stabilization under Hypoxia In Vitro but Does Not Affect Tumorigenesis in Multiple Breast Cancer Models In Vivo

Intratumoral hypoxia is a poor prognostic factor associated with reduced disease-free survival in many cancer types, including breast cancer. Hypoxia encourages tumor cell proliferation, stimulates angiogenesis and lymphangiogenesis, and promotes epithelial-mesenchymal transition and metastasis. Tumor cells respond to a hypoxic state by stabilizing the Hif-1α subunit of the Hypoxia-Inducible Factor (HIF) transcription factor to promote expression of various tumor- and metastasis-promoting hypoxic response genes. The antioxidant N-acetylcysteine (NAC) was recently shown to prevent Hif-1α stabilization under hypoxia, and has been identified as a potential alternative method to target the hypoxic response in tumors. We utilized three orthotopic syngeneic murine models of breast cancer, the PyMT, EO771 and 4T1.2 models, to investigate the ability of NAC to modulate the hypoxic response in vitro and in vivo. While NAC prevented Hif-1α stabilization under hypoxia in vitro and increased levels of glutathione in the blood of mice in vivo, this did not translate to a difference in tumor growth or the hypoxic state of the tumor compared to untreated control mice. In addition, NAC treatment actually increased metastatic burden in an experimental metastasis model. This work raises questions regarding the validity of NAC as an anti-tumorigenic agent in breast cancer, and highlights the need to further investigate its properties in vivo in different cancer models.

Siah2-deficient mice show impaired skin wound repair

Hypoxia is associated with the dermal wound healing process and hypoxia signaling is presumed to be crucial for normal wound repair. The Siah2 ubiquitin ligase controls the abundance of hypoxia‐inducible factor‐1 alpha, and loss of Siah2 results in destabilization of hypoxia‐inducible factor‐1 alpha under hypoxia. Utilizing Siah2−/− mice we demonstrate that cutaneous wound healing is impaired in these mice. Wounds in Siah2−/− mice heal slower and are associated with delayed induction of myofibroblast infiltration and reduced collagen deposition. This coincides with delayed angiogenesis and reduced macrophage infiltration into the wounds of Siah2−/− mice. We furthermore demonstrate that primary Siah2−/− dermal fibroblasts have reduced migratory capacities and produce less collagen than wild‐type fibroblasts. Additionally, Siah2−/− fibroblasts showed conserved responses to transforming growth factor‐β at the receptor level (pSmad 2C activation) but reduced responses downstream. Together, our data show, for the first time, that Siah2 is involved as a positive regulator in the wound healing response. Understanding the role of hypoxia signaling in tissue repair and fibrosis and interference with the hypoxia signaling pathway via regulation of Siah2 may provide new targets for clinical regulation of fibrosis and scarring.

Loss of Siah2 does not impact angiogenic potential of murine endothelial cells

Angiogenesis is triggered in response to hypoxia under many circumstances, from healthy cells and tissues during embryogenesis to pathological conditions like the formation of new blood vessels to supply tumours and promote invasive cancer. Siah2 has been shown to regulate the hypoxia pathway upstream of hypoxia-induced transcription factor subunit Hif-1alpha, and therefore may play an important role in angiogenesis in response to hypoxic stress in endothelial cells. This study aims to investigate the basic function of Siah2 in endothelial cells under hypoxia and to test the ability of Siah2 deficient cells to mount an angiogenic response when deprived of oxygen. We and others have previously shown that Siah2 is crucial for mediating the hypoxic response in many different cell types studied. In this study however, we describe that Siah2(-/-) endothelial cells have an intact hypoxic signalling pathway, including Hif-1alpha stabilisation and gene expression, the first report of a tissue or cell lineage in which the loss of Siah2 does not seem to impact hypoxic response signalling. In mice, the infiltration of Siah2(-/-) endothelial cells into a Matrigel plug containing a VEGF-A attractant was similar compared with wildtype endothelial cells. Ex vivo however, there was a reduced capacity of Siah2(-/-) aorta to form tubes or new vessels. Thus, we conclude that Siah2 is not essential for the hypoxic response of endothelial cells.