Alexes C. Daquinag

White Adipose Tissue Cells Are Recruited by Experimental Tumors and Promote Cancer Progression in Mouse Models

The connection between obesity and accelerated cancer progression has been established, but the mediating mechanisms are not well understood. We have shown that stromal cells from white adipose tissue (WAT) cooperate with the endothelium to promote blood vessel formation through the secretion of soluble trophic factors. Here, we hypothesize that WAT directly mediates cancer progression by serving as a source of cells that migrate to tumors and promote neovascularization. To test this hypothesis, we have evaluated the recruitment of WAT-derived cells by tumors and the effect of their engraftment on tumor growth by integrating a transgenic mouse strain engineered for expansion of traceable cells with established allograft and xenograft cancer models. Our studies show that entry of adipose stromal and endothelial cells into systemic circulation leads to their homing to and engraftment into tumor stroma and vasculature, respectively. We show that recruitment of adipose stromal cells by tumors is sufficient to promote tumor growth. Finally, we show that migration of stromal and vascular progenitor cells from WAT grafts to tumors is also associated with acceleration of cancer progression. These results provide a biological insight for the clinical association between obesity and cancer, thus outlining potential avenues for preventive and therapeutic strategies.

Stromal Progenitor Cells from Endogenous Adipose Tissue Contribute to Pericytes and Adipocytes That Populate the Tumor Microenvironment

Epidemiologic studies associate cancer with obesity, but the pathophysiologic connections remain obscure. In this study, we show that obesity facilitates tumor growth in mice irrespective of concurrent diet, suggesting a direct effect of excess white adipose tissue (WAT). When transplanted into mice, adipose stromal cells (ASC) can serve as perivascular adipocyte progenitors that promote tumor growth, perhaps helping explain the obesity-cancer link. In developing this hypothesis, we showed that ASCs are expanded in obesity and that they traffic from endogenous WAT to tumors in several mouse models of cancer. Strikingly, a comparison of circulating and tumor-infiltrating cell populations in lean, and obese mice revealed that cancer induces a six-fold increase of ASC frequency in the systemic circulation. We obtained evidence that ASCs mobilized in this way can be recruited into tumors, where they can be incorporated into blood vessels as pericytes and they can differentiate into adipocytes in an obesity-dependent manner. Extending this evidence, we found that increased tumor vascularization (reflected by changes in tumor vascular morphology and a two-fold increase in vascular density) was associated with intratumoral adipocytes and elevated proliferation of neighboring malignant cells. Taken together, our results suggest that ASCs recruited from endogenous adipose tissue can be recruited by tumors to potentiate the supportive properties of the tumor microenvironment.

The Yeast PH Domain Proteins Slm1 and Slm2 Are Targets of Sphingolipid Signaling during the Response to Heat Stress

ABSTRACT The PH domain-containing proteins Slm1 and Slm2 were previously identified as effectors of the phosphatidylinositol-4,5-bisphosphate (PI4,5P2) and TORC2 signaling pathways. Here, we demonstrate that Slm1 and Slm2 are also targets of sphingolipid signaling during the heat shock response. We show that upon depletion of cellular sphingolipid levels, Slm1 function becomes essential for survival under heat stress. We further demonstrate that Slm proteins are regulated by a phosphorylation/dephosphorylation cycle involving the sphingolipid-activated protein kinases Pkh1 and Pkh2 and the calcium/calmodulin-dependent protein phosphatase calcineurin. By using a combination of mass spectrometry and mutational analysis, we identified serine residue 659 in Slm1 as a site of phosphorylation. Characterization of Slm1 mutants that mimic dephosphorylated and phosphorylated states demonstrated that phosphorylation at serine 659 is vital for survival under heat stress and promotes the proper polarization of the actin cytoskeleton. Finally, we present evidence that Slm proteins are also required for the trafficking of the raft-associated arginine permease Can1 to the plasma membrane, a process that requires sphingolipid synthesis and actin polymerization. Together with previous work, our findings suggest that Slm proteins are subject to regulation by multiple signals, including PI4,5P2, TORC2, and sphingolipids, and may thus integrate inputs from different signaling pathways to temporally and spatially control actin polarization.

Vascular targeting of adipose tissue as an anti-obesity approach

Development of obesity is characterized by hypertrophy and hyperplasia of adipocytes in white adipose tissue (WAT). This process relies on concomitant angiogenesis. Results from experimental inhibition or depletion of cells comprising the vasculature in animal models have contributed to the understanding of the mechanisms governing expansion of WAT. Disruption of neovascularization might be potentially useful for obesity prevention. In addition, approaches in which the mature WAT vasculature is disrupted have been sought with the aim of combating obesity after its onset. Other cell types in WAT, including adipose stromal cells, which support angiogenesis, could represent alternative targets for combinatorial WAT treatment. This review discusses recent advances in WAT vascular targeting and implications for the development of new anti-obesity therapeutics.

Type I Phosphatidylinositol Phosphate Kinase Beta Regulates Focal Adhesion Disassembly by Promoting β1 Integrin Endocytosis

ABSTRACT Cell migration requires the regulated disassembly of focal adhesions, but the underlying mechanisms remain poorly defined. We have previously shown that focal adhesion disassembly requires the dynamin 2- and clathrin-dependent endocytosis of ligand-activated β1 integrins. Here, we identify type I phosphatidylinositol phosphate kinase beta (PIPKIβ), an enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI4,5P2), as a key regulator of this process. We found that knockdown of PIPKIβ by RNA interference blocks the internalization of active β1 integrins and impairs focal adhesion turnover and cell migration. These defects are caused by the failure to target the endocytic machinery, including clathrin adaptors and dynamin 2, to focal adhesion sites. As a consequence, depletion of PIPKIβ blocks clathrin assembly at adhesion plaques and prevents complex formation between dynamin 2 and focal adhesion kinase (FAK), a critical step in focal adhesion turnover. Together, our findings identify PIPKIβ as a novel regulator of focal adhesion disassembly and suggest that PIPKIβ spatially regulates integrin endocytosis at adhesion sites to control cell migration.

A peptide probe for targeted brown adipose tissue imaging

The presence of brown adipose tissue (BAT) responsible for thermogenic energy dissipation has been revealed in adult humans and has high clinical importance. Due to limitations of current methods for BAT detection, analyzing the abundance and localization of BAT in the body has remained challenging. Here, we screen a combinatorial peptide library in mice and characterize a peptide (with the sequence CPATAERPC) that selectively binds to the vascular endothelium of BAT, but not of intraperitoneal white adipose tissue (WAT). We show that in addition to BAT, this peptide probe also recognizes the vasculature of BAT-like depots of subcutaneous WAT. Our results indicate that the CPATAERPC peptide localizes to BAT even in the absence of sympathetic nervous system stimulation. Finally, we demonstrate that this probe can be used to identify BAT depots in mice by whole body near-infrared (NIR) fluorescence imaging.

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.

Targeted Proapoptotic Peptides Depleting Adipose Stromal Cells Inhibit Tumor Growth

Progression of many cancers is associated with tumor infiltration by mesenchymal stromal cells (MSC). Adipose stromal cells (ASC) are MSC that serve as adipocyte progenitors and endothelium-supporting cells in white adipose tissue (WAT). Clinical and animal model studies indicate that ASC mobilized from WAT are recruited by tumors. Direct evidence for ASC function in tumor microenvironment has been lacking due to unavailability of approaches to specifically inactivate these cells. Here, we investigate the effects of a proteolysis-resistant targeted hunter-killer peptide D-WAT composed of a cyclic domain CSWKYWFGEC homing to ASC and of a proapoptotic domain KLAKLAK2. Using mouse bone marrow transplantation models, we show that D-WAT treatment specifically depletes tumor stromal and perivascular cells without directly killing malignant cells or tumor-infiltrating leukocytes. In several mouse carcinoma models, targeted ASC cytoablation reduced tumor vascularity and cell proliferation resulting in hemorrhaging, necrosis, and suppressed tumor growth. We also validated a D-WAT derivative with a proapoptotic domain KFAKFAK2 that was found to have an improved cytoablative activity. Our results for the first time demonstrate that ASC, recruited as a component of tumor microenvironment, support cancer progression. We propose that drugs targeting ASC can be developed as a combination therapy complementing conventional cancer treatments.

Non-glycanated Decorin Is a Drug Target on Human Adipose Stromal Cells

Adipose stromal cells (ASCs) have been identified as a mesenchymal cell population recruited from white adipose tissue (WAT) by tumors and supporting cancer progression. We have previously reported the existence of a non-glycanated decorin isoform (ngDCN) marking mouse ASCs. We identified a peptide CSWKYWFGEC that binds to ngDCN and hence can serve as a vehicle for ASC-directed therapy delivery. We used hunter-killer peptides composed of CSWKYWFGEC and a pro-apoptotic moiety to deplete ASCs and suppress growth of mouse tumors. Here, we report the discovery of the human non-glycanated decorin isoform. We show that CSWKYWFGEC can be used as a probe to identify ASCs in human WAT and tumors. We demonstrate that human ngDCN is expressed on ASC surface. Finally, we validate ngDCN as a molecular target for pharmacological depletion of human ASCs with hunter-killer peptides. We propose that ngDCN-targeting agents could be developed for obesity and cancer treatment.

PDGFRα/PDGFRβ signaling balance modulates progenitor cell differentiation into white and beige adipocytes

The relative abundance of thermogenic beige adipocytes and lipid-storing white adipocytes in adipose tissue underlie its metabolic activity. The roles of adipocyte progenitor cells, which express PDGFRα or PDGFRβ, in adipose tissue function have remained unclear. Here, by defining the developmental timing of PDGFRα and PDGFRβ expression in mouse subcutaneous and visceral adipose depots, we uncover depot specificity of pre-adipocyte delineation. We demonstrate that PDGFRα expression precedes PDGFRβ expression in all subcutaneous but in only a fraction of visceral adipose stromal cells. We show that high-fat diet feeding or thermoneutrality in early postnatal development can induce PDGFRβ+ lineage recruitment to generate white adipocytes. In contrast, the contribution of PDGFRβ+ lineage to beige adipocytes is minimal. We provide evidence that human adipose tissue also contains distinct progenitor populations differentiating into beige or white adipocytes, depending on PDGFRβ expression. Based on PDGFRα or PDGFRβ deletion and ectopic expression experiments, we conclude that the PDGFRα/PDGFRβ signaling balance determines progenitor commitment to beige (PDGFRα) or white (PDGFRβ) adipogenesis. Our study suggests that adipocyte lineage specification and metabolism can be modulated through PDGFR signaling. Summary: White and beige adipocytes arise from distinct progenitor cell lineages. In both mice and humans, beige and white adipogenesis is induced by PDGFRα and PDGFRβ signaling, respectively.