Tumorigenic potential is restored during differentiation in fusion-reprogrammed cancer cells

Detailed understanding of the mechanistic steps underlying tumor initiation and malignant progression is critical for insights of potentially novel therapeutic modalities. Cellular reprogramming is an approach of particular interest because it can provide a means to reset the differentiation state of the cancer cells and to revert these cells to a state of non-malignancy. Here, we investigated the relationship between cellular differentiation and malignant progression by the fusion of four independent mouse cancer cell lines from different tissues, each with differing developmental potentials, to pluripotent mouse embryonic stem (ES) cells. Fusion was accompanied by loss of differentiated properties of the four parental cancer cell lines and concomitant emergence of pluripotency, demonstrating the feasibility to reprogram the malignant and differentiative properties of cancer cells. However, the original malignant and differentiative phenotypes re-emerge upon withdrawal of the fused cells from the embryonic environment in which they were maintained. cDNA array analysis of the malignant hepatoma progression implicated a role for Foxa1, and silencing Foxa1 prevented the re-emergence of malignant and differentiation-associated gene expression. Our findings support the hypothesis that tumor progression results from deregulation of stem cells, and our approach provides a strategy to analyze possible mechanisms in the cancer initiation.Investigations into cancer formation are most often focused on the accumulation of specific genetic and epigenetic alterations that alter the expression of the oncogenes and tumor suppressors regulating cell cycle, apoptosis, DNA repair, cell adhesion and signaling.^{1, 2, 3} Less often considered, the tumorigenic process can also be regarded from a standpoint of a dynamic relationship between malignant progression and cellular differentiation.⁴ During the course of development, normal stem cells differentiate into specific types of cells by exchanging and interpreting signaling molecules with the surrounding microenvironment. Accumulating evidence indicates that cancer cells may also release and receive cues from the surroundings that contribute to malignant progression.⁵ However, how tumor cell–niche interactions drive malignancy remains a critical gap in our overall understanding of the cancer process, and understanding this process has significant potential in providing new prognosis strategy for therapeutic intervention at early stages of cancer development.Reprogramming can alter differentiation properties of adult cells, and this approach may be exploitable to reverse the malignant programming in cancer cells.⁶ Published reports documented the use of nuclear transfer by implanting the nuclei of mouse melanoma,^{7, 8} embryonic carcinoma⁸ and medulloblastoma⁹ into mouse oocytes. Although the nuclear transferred cells regained pluripotent potential, the malignant properties remained, indicating incomplete reprogramming in reproductive and therapeutic cloning with this approach.^{10, 11} Separately, defined factors OSMK (Oct4, Sox2, c-Myc and Klf4) were tested for the ability to reprogram both solid and liquid malignant tumors including chronic myeloid leukemia,^{12, 13} gastrointestinal cancer,¹⁴ melanoma¹⁵ and sarcoma cells.^{16, 17} Using the OSMK approach, late-stage cancer cells could revert back to an earlier state, bolstering enthusiasm for the discovery of new insights in cancer initiation and progression. However, OSKM-reprogrammed cells had limited pluripotency and altered tumorigenic potential during re-dedifferentiation. Moreover, the OSKM approach to promote pluripotency was effective only on a limited subset of cancer types.¹⁸ The shortcomings of OSMK may be due to the presence of oncongenic factors (c-Myc and Klf4) or to the intrinsic defects of the strategy.^{19, 20} Most importantly, these shortcomings hinder the use of OSKM approach to investigate tumor progression in reprogrammed cancer cells.ES cell-induced fusion provides a more efficient and effective reprogramming strategy to test the reversibility of tumorigenic potential. In previous studies using normal adult cells, the normal cell fusion hybrids exhibited epigenetic characteristics similar to ES cells, such as reactivation of histone modifications and a DNA hypomethylation state within the Oct4 promoter.^{21, 22, 23, 24, 25, 26, 27, 28, 29} We generated a fusion hybrid of mouse hepatoma cells and mouse embryonic stem (ES) cells previously.³⁰ The resultant ES-Hepa hybrids forfeited tumorigenic properties, but the forfeiture was reversible and tumorigenic properties re-emerge upon removal of the cells from embryonic environments. We observed that H3K27 trimethylation, which was independent of H3K9 dimethylation, was an early event in the silencing of p16^INK4a during re-emergence of the tumorigenic profile, a finding that was supported by a number of other groups studying the progression mechanisms of hepatocellular carcinoma (HCC).^{30, 31, 32} These previous research highlighted the remarkable developmental plasticity of HCC during cancer progression and engendered two important questions. First, is developmental plasticity a ubiquitous phenomenon in all cancer progression? Second, how does lineage specification relate to cancer progression? While the reprogramming approach holds significant promise for future cancer therapies, current data also caution that partial or incomplete reprogramming can lead to a worse outcome by inducing more invasive phenotypes.³³ Clearly, much critical knowledge remains to be learned regarding the association between the differentiation and tumorigenic phenotypes in cellular reprogramming and the molecular events driving these events.Here, we present data showing that four cancer cell lines, each endowed with distinct lineage-differentiated characteristics, were reprogrammed by fusion with ES cells to yield ES-cancer hybrids with characteristics similar to pluripotent ES cells with diminished tumorigenic gene expression patterns. We observed that the sustainability of pluripotent ES characteristics was dependent on environmental cues. Removal of cells from embryonic culturing conditions resulted in the re-emergence of the original differentiated and tumorigenic gene expression patterns. cDNA array and RT-PCR data in malignant hepatoma progression discovered Foxa1 as a potential checkpoint regulator, affecting a network of 224 genes that might be participants for the differentiation and malignant progression. Silencing of Foxa1 inhibited the re-emergence of the malignant and differentiated pattern of gene expression. Our observations suggest that cell fusion-mediated reprogramming is a novel and potentially useful strategy for the identification and testing of molecules involved in cancer initiation and progression, with exploitable modalities for new cancer therapies.     

Enhancing mammary differentiation by overcoming lineage-specific epigenetic modification and signature gene expression of fibroblast-derived iPSCs

Recent studies have shown that induced pluripotent stem cells (iPSCs) retain a memory of their origin and exhibit biased differentiation potential. This finding reveals a severe limitation in the application of iPSCs to cell-based therapy because it means that certain cell types are not available for reprogramming for patients. Here we show that the iPSC differentiation process is accompanied by profound gene expression and epigenetic modifications that reflect cells'' origins. Under typical conditions for mammary differentiation, iPSCs reprogrammed from tail-tip fibroblasts (TF-iPSCs) activated a fibroblast-specific signature that was not compatible with mammary differentiation. Strikingly, under optimized conditions, including coculture with iPSCs derived from the mammary epithelium or in the presence of pregnancy hormones, the fibroblast-specific signature of TF-iPSCs obtained during differentiation was erased and cells displayed a mammary-specific signature with a markedly enhanced ability for mammary differentiation. These findings provide new insights into the precise control of differentiation conditions that may have applications in personalized cell-based therapy.The mammary gland is a primary target for carcinogenesis. Breast cancer occurs at a high rate and affects one in eight women in Western countries during their lifetime.^{1, 2} In the United States alone, 232 340 new invasive breast cancer cases were reported for women in 2013 and 39 620 patients died.³ Regenerative therapy of the damaged mammary gland tissues is the best way to restore breast functions; therefore, the creation of stem cells that are capable of developing into fully functional mammary glands is desirable. There are two distinct types of pluripotent stem cells that may be used for this purpose. The first is embryonic stem cells (ESCs) derived from the inner cell mass of embryonic blastocysts,⁴ and the second is induced pluripotent stem cells (iPSCs) obtained by reprogramming somatic cells.⁵ Although, in theory, both ESCs and iPSCs can be differentiated into any type of mature cell, use of the latter is more desirable because it does not require the killing of embryos, and the cells can be derived from virtually any type of tissue. In addition, because iPSCs can be generated from the same patient, the use of iPSCs avoids the immunosuppressive reactions that have long hampered organ and tissue transplantation.^{6, 7, 8} However, recent studies have shown that some iPSCs seem to retain a memory of their origin and exhibit skewed potential during differentiation for tissue/organ formation.^{9, 10, 11, 12, 13, 14} This feature may represent a limitation if certain cell types from diseased tissues or organs are not available for reprogramming.Numerous studies about the use of ESCs have indicated that, although these cells have the potential to generate all cell types, their differentiation depends critically on many factors.^{14, 15, 16} Precise conditions are required for driving cells into specific pathways leading to new lineage formation (reviewed in Murry and Keller¹⁷ and Cahan and Daley¹⁸). Based on these observations, we hypothesized that the skewed differentiation of iPSCs could be overcome by providing favorable conditions for differentiation. To test this hypothesis, we have generated iPSCs from mouse mammary epithelial cells (ME-iPSCs) and mouse-tail fibroblasts (TF-iPSCs), and have studied the gene expression profiles and epigenetic modifications during differentiation. We found that, although these iPSCs activate distinct signature memories that are reflective of their origins during the differentiation process, the fate of iPSCs could be redirected under optimized conditions in favor of the formation of a desired tissue/organ.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

Nitric oxide is a positive regulator of the Warburg effect in ovarian cancer cells

Ovarian cancer (OVCA) is among the most lethal gynecological cancers leading to high mortality rates among women. Increasing evidence indicate that cancer cells undergo metabolic transformation during tumorigenesis and growth through nutrients and growth factors available in tumor microenvironment. This altered metabolic rewiring further enhances tumor progression. Recent studies have begun to unravel the role of amino acids in the tumor microenvironment on the proliferation of cancer cells. One critically important, yet often overlooked, component to tumor growth is the metabolic reprogramming of nitric oxide (NO) pathways in cancer cells. Multiple lines of evidence support the link between NO and tumor growth in some cancers, including pancreas, breast and ovarian. However, the multifaceted role of NO in the metabolism of OVCA is unclear and direct demonstration of NO''s role in modulating OVCA cells'' metabolism is lacking. This study aims at indentifying the mechanistic links between NO and OVCA metabolism. We uncover a role of NO in modulating OVCA metabolism: NO positively regulates the Warburg effect, which postulates increased glycolysis along with reduced mitochondrial activity under aerobic conditions in cancer cells. Through both NO synthesis inhibition (using L-arginine deprivation, arginine is a substrate for NO synthase (NOS), which catalyzes NO synthesis; using L-Name, a NOS inhibitor) and NO donor (using DETA-NONOate) analysis, we show that NO not only positively regulates tumor growth but also inhibits mitochondrial respiration in OVCA cells, shifting these cells towards glycolysis to maintain their ATP production. Additionally, NO led to an increase in TCA cycle flux and glutaminolysis, suggesting that NO decreases ROS levels by increasing NADPH and glutathione levels. Our results place NO as a central player in the metabolism of OVCA cells. Understanding the effects of NO on cancer cell metabolism can lead to the development of NO targeting drugs for OVCAs.Despite recent medical and pharmaceutical advances in cancer research, ovarian cancer (OVCA) remains one of the most deadly gynecological malignancies, with most of the cancer first detected in late stages when metastasis has already occurred.¹ Only 20% of OVCA patients are diagnosed when cancer has not spread past the ovaries; in the other 80% of cases, the cancer has metastasized, most frequently to the peritoneum.² Platinum-based preoperative chemotherapy is the standard of care of early stage disease, and surgical resection along with platinum-based postoperative chemotherapy is the standard of care for late stage disease.¹ However, many platinum-based chemotherapy drugs come with unwanted side effects. Therefore, an alternative therapy for OVCA is needed.Nitric oxide (NO) shows promise either as a cancer therapeutic agent by itself or as a target of cancer therapies.³ This may be because NO can act as a signaling molecule or as a source of oxidative and nitrosative stress.⁴ NO can stimulate mitochondrial biogenesis through PGC-1-related coactivator⁵ and increase mitochondrial function.^{6, 7} In follicular thyroid carcinoma cells, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, was shown to increase the expression of genes involved in mitochondrial biogenesis.^{8, 9} A 14-day treatment of lung carcinoma cells with dipropylenetriamine NONOate (DETA-NONOate), another NO donor, increased cell migration compared with the absence of treatment.¹⁰ In breast cancer cells, exogenous NO increased cell proliferation, as well as cyclin-D1 and ornithine decarboxylase expression.¹¹ In prostate cancer cells, NO was shown to inhibit androgen receptor-dependent promoter activity and proliferation of androgen-dependent cells, indicating that NO would select for the development of prostate cancer cells that are androgen-independent.¹² NO has even been shown to inhibit mitochondrial ATP production, and therefore inhibit apoptosis, as ATP is necessary for the apoptotic process.¹³ Moreover, inducible nitric oxide synthase (iNOS) knockout mice had less tumor formation than wild-type mice, indicating that NO promotes lung tumorigenesis.¹⁴ On the other hand, NO production, as induced by proinflammatory cytokines, induced apoptosis in OVCA cells.³ NOS overexpression by transfection of a plasmid containing NOS-3 DNA resulted in increased cell death in HepG2 cells.¹⁵ In another study, NO was implicated in N-(4-hydroxyphenyl) retinamide-mediated apoptosis.¹⁶ Finally, iNOS expression in p53-depleted mice increased apoptosis of lymphoma cells compared with p53-deficient mice without iNOS expression.¹⁷ Therefore, NO has been seen to have both an anti-tumorigenic as well as a pro-tumorigenic effect.Arginine, a conditionally essential amino acid used to produce NO, is also a potential target for cancer therapy. L-arginine is normally produced by the body; however, in some diseased states, more arginine than what the body normally produces is required.¹⁸ Arginine sources include protein breakdown or directly from the diet, in addition to de novo synthesis.¹⁹ In the de novo production of L-arginine, citrulline and aspartate are first converted to argininosuccinate by arginase, which is then split into arginine and fumarate by argininosuccinate lyase.²⁰ L-arginine can also be converted to citrulline and NO through NO synthase (NOS).¹⁹ Some cancer cells, including melanoma and hepatocellular carcinoma, do not express argininosuccinate synthase (ASS), an enzyme involved in arginine production and thus rely on exogenous arginine.¹⁹ For these cancers, arginine-deprivation therapy is being heavily explored as a treatment.^{21, 22} OVCA cells have been shown to express ASS.²³ In fact, OVCA cells were shown to have increased expression of ASS compared with normal ovarian surface epithelium.²⁴ As OVCA can synthesize arginine de novo, strategies which target arginine''s conversion into citrulline are needed for regulating OVCA tumor growth.Recent studies suggest that cancer cells undergo metabolic reprogramming, which drives cancer cells'' growth and progression.^{25, 26, 27, 28, 29, 30, 31, 32, 33} One critically important, yet often overlooked, component to tumor growth is the metabolic rewiring of NO pathways in OVCA cells. Despite considerable investigation on NO''s regulation of cancer cell proliferation and growth, mechanistic details regarding the effect of NO on cancer cell metabolism is still lacking: specifically, how NO affects glycolysis, TCA cycle flux, and ROS production. Studies on the effects of NO on cancer cell metabolism have mainly focused on the effect of NO on mitochondrial respiration.^{34, 35, 36, 37} NO has been shown to inhibit cytochrome c oxidase (COX) in the mitochondria of breast cancer cells, as well as decrease oxygen consumption rate.^{37, 38, 39} Moncada and colleagues studied the effect of NO on the metabolism of rat cortical astrocytes and neurons, two cells with different glycolytic capacities. They showed that NO decreased ATP concentration, which led to an increase in glycolysis in astrocytes, but not in neurons, indicating that glycolytic capacity affects the metabolic response of these cells to NO.⁴⁰ NO was shown to reduce ATP production via OXPHOS in rat reticulocytes, cells that produce 90% of their ATP from OXPHOS.⁴¹ Endothelial NOS (eNOS) was shown to have a role in the upregulation of GLUT4 transporters by AMPK and AICAR in the heart muscle.⁴² Additionally, NO can serve to stabilize HIF-1α in hypoxic conditions through S-nitrosylation of PHD2,⁴ and as HIF-1α upregulates GLUT transporters and glycolysis,⁴³ NO may affect the metabolism of cancer cells.Although NO is found to affect glycolysis of normal cells, how NO modulates glycolysis of OVCA cells is less understood. The multifaceted role of NO in the metabolism of OVCA is unclear, and direct demonstration of NO''s role in modulating the metabolism of OVCA cells is lacking. This study aims at understanding the mechanistic links between NO and the overall cancer metabolism – specifically, its effects on glycolysis, TCA cycle, OXPHOS, and ROS production – of OVCA cells. Our results show that NO decreases mitochondrial respiration, forcing OVCA cells to undergo higher glycolytic rates to maintain ATP production levels. Our work is the first to illustrate the central role of NO on OVCA metabolism – specifically, showing how NO (i) positively regulates the Warburg effect in OVCA cell, (ii) maintains low ROS levels by upregulating NADPH generation, and (ii) negatively alters mitochondrial respiration, thus promoting cancer growth and proliferation. Our work is also unique in that it is the first to explore the effects of NO on TCA cycle flux and glutaminolysis, potentially also affecting ROS levels by affecting antioxidant levels. In conclusion, by elucidating the effects of NO on cancer metabolism and ROS levels, we have a better understanding of the different mechanisms by which NO affects cancer cell growth. This understanding may lead to potentially useful therapies to halt cancer progression.     

Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization.Many diseases, including rheumatoid arthritis, pulmonary fibrosis, adult respiratory distress syndrome, and inflammatory bowel disease,^{1, 2, 3, 4} are commonly marked by impaired resolution of inflammation that is linked to defects in the phagocytic clearance of apoptotic cells.^{5, 6, 7} Apoptotic cell (AC) clearance normally eliminates a plethora of pro-inflammatory stimuli,^{8, 9} and the recognition of ACs by phagocytes¹⁰ limits progression to necrosis,¹¹ suppresses pro-inflammatory mediator production, and induces IL-10 and TGF-β release.^{12, 13} As defective clearance of ACs is associated with the development of inflammatory disease and autoimmunity,^{14, 15} new therapeutic approaches designed to increase the capacity of phagocytes to remove ACs could effectively promote the resolution of inflammation.Phagocytosis of ACs can be regulated by soluble mediators, including cytokines,^{16, 17} prostaglandins and lipoxins,^{17, 18, 19} serum proteins,²⁰ agonists of Liver X receptors (LXRs),^{17, 21} and glucocorticoids (GC).^{17, 22} In particular, LXR agonists and GCs promote phagocytosis of ACs predominantly via a Tyro3/Axl/Mer (TAM) receptor tyrosine kinase (RTK)-dependent pathway.^{17, 21, 23} There are two established ligands for the TAM RTKs, Protein S (gene name Pros1), which activates Tyro3 and Mer, and Gas6, which activates all three TAMs,^{24, 25} although other ligands have been suggested.^{26, 27} The amino terminal Gla domains of Protein S and Gas6 bind to phosphatidylserine (PtdSer) on the plasma membrane of ACs,²⁸ a potent ‘eat-me'' signal by which ACs are recognized by phagocytes.²⁹ TAM receptors bind to the carboxy terminal domains of Protein S and Gas6, which effectively act as molecular ‘bridges'' between PtdSer on the AC and TAM receptors on the phagocyte.^{17, 30, 31} TAM receptor- and ligand-deficient mice exhibit defective phagocytic pruning of photoreceptor outer segments by retinal pigment epithelial (RPE) cells of the eye,^{32, 33, 34} defective clearance of apoptotic germ cells by Sertoli cells of the testis,³⁵ and defective clearance of ACs by macrophages/dendritic cells in lymphoid organs.³⁶ These phenotypes are also detectable in Mer (gene name Mertk) single knockouts.³⁷ In addition to phagocytic clearance, TAM signaling also has a pivotal role in controlling the innate immune response to pathogenic stimuli.^{13, 17, 38}Although the importance of Mer in the internalization of ACs by macrophages is now well-established, this receptor has been thought not to have a significant role in the initial ‘tethering'' of ACs to the macrophage surface.^{36, 39} In their studies, Scott et al.³⁶ used peritoneal macrophages for which tethering of ACs has now been shown to be mediated by T-cell immunoglobulin and mucin domain-containing molecule 4 (TIM4).³⁹ Subsequent internalization of tethered ACs is then mediated by either integrin αvβ3- or Mer-mediated signaling.^{39, 40} Similarly, for RPE cells, the initial capture of photoreceptor outer segments by RPE cells required the integrin αvβ5,⁴¹ with Mer-dependent signaling necessary for subsequent internalization. To further probe the mechanistic role of Mer in AC recognition and engulfment, we have now examined macrophages that predominantly use a Mer-dependent AC phagocytosis mechanism.^{17, 23} We show that in these cells, which do not express TIM4, Mer has the capacity to serve a unique dual role in mediating both tethering of ACs to the macrophage surface as well as subsequent AC engulfment.     

Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner

Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.¹ Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.^{2, 3, 4} Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.^{2, 5, 6, 7} Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.^{8, 9, 10, 11, 12} Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.^{8, 11, 12} Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.^{8, 9} Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,¹³ liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.^{12, 14, 15}Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon^{16, 17} and prostate cancers,^{18, 19} contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis²⁰ and is necessary for tBid-induced cancer cell apoptosis.²¹ Loss of Bax has been reported to promote tumor development in animal models.²² Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.^{23, 24, 25} p53 has been reported to be a tumor suppressor,²⁶ and its mutant can cause chemoresistance in cancer cells.^{27, 28, 29} Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.^{30, 31} Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.³² Some studies have revealed that Bak is a determinant of cancer cell apoptosis.^{33, 34} Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.^{33, 35} In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells.     

Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells

Tumor heterogeneity is in part determined by the existence of cancer stem cells (CSCs) and more differentiated tumor cells. CSCs are considered to be the tumorigenic root of cancers and suggested to be chemotherapy resistant. Here we exploited an assay that allowed us to measure chemotherapy-induced cell death in CSCs and differentiated tumor cells simultaneously. This confirmed that CSCs are selectively resistant to conventional chemotherapy, which we revealed is determined by decreased mitochondrial priming. In agreement, lowering the anti-apoptotic threshold using ABT-737 and WEHI-539 was sufficient to enhance chemotherapy efficacy, whereas ABT-199 failed to sensitize CSCs. Our data therefore point to a crucial role of BCLXL in protecting CSCs from chemotherapy and suggest that BH3 mimetics, in combination with chemotherapy, can be an efficient way to target chemotherapy-resistant CSCs.Colorectal cancer is the third most common cancer worldwide.^{1, 2} Patients with advanced stage colorectal cancer are routinely treated with 5-fluorouracil (5-FU), leucovorin and oxaliplatin (FOLFOX), or with 5-FU, leucovorin and irinotecan (FOLFIRI), often in combination with targeted agents such as anti-VEGF or anti-EGFR at metastatic disease.^{3, 4, 5, 6} Despite this intensive treatment, therapy is still insufficiently effective and chemotherapy resistance occurs frequently. Although still speculative, it has been suggested that unequal sensitivity to chemotherapy is due to an intratumoral heterogeneity that is orchestrated by cancer stem cells (CSCs) that can self-renew and give rise to more differentiated progeny.^{7, 8} When isolated from patients, CSCs efficiently form tumors upon xenotransplantation into mice which resemble the primary tumor from which they originated.^{9, 10, 11} In addition, many xenotransplantation studies have emphasized the importance of CSCs for tumor growth.^{9, 10, 11, 12} Colon CSCs were originally isolated from primary human colorectal tumor specimens using CD133 as cell surface marker and shown to be highly tumorigenic when compared with the non-CSCs population within a tumor.^{9, 10} Later, other cell surface markers as well as the activity of the Wnt pathway have been used to isolate colon CSCs from tumors.^{12, 13} Spheroid cultures have been established from human primary colorectal tumors that selectively enrich for the growth of colon CSCs,^{11, 12} although it is important to realize that these spheres also contain more differentiated tumor cells.¹² In agreement, we have shown that the Wnt activity reporter that directs the expression of enhanced green fluorescent protein (TOP-GFP) allows for the separation of CSCs from more differentiated progeny in the spheroid cultures.¹²CSCs are suggested to be responsible for tumor recurrence after initial therapy, as they are considered to be selectively resistant to therapy.^{11, 14} Conventional chemotherapy induces, among others, DNA damage and subsequent activation of the mitochondrial cell death pathway, which is regulated by a balance between pro- and anti-apoptotic BCL2 family members.¹⁵ Upon activation of apoptosis, pro-apoptotic BH3 molecules are activated and these may perturb the balance in favor of apoptosis initiated by mitochondrial outer membrane polarization (MOMP), release of cytochrome c and subsequent activation of a caspase cascade.The apoptotic balance of cancer cells can be measured with the use of a functional assay called BH3 profiling.¹⁶ BH3 profiling is a method to determine the apoptotic ‘priming'' level of a cell by exposing mitochondria to standardized amounts of roughly 20-mer peptides derived from the alpha-helical BH3 domains of BH3-only proteins and determining the rate of mitochondrial depolarization. Using this approach, priming was measured in various cancers and compared with normal tissues.^{17, 18} In all cancer types tested, the mitochondrial priming correlated well with the observed clinical response to chemotherapy. That is, cancers that are highly primed are more chemosensitive, whereas chemoresistant tumor cells and normal tissues are poorly primed.^{17, 18} This suggests that increasing mitochondrial priming can potentially increase chemosensitivity, which can be achieved by directly inhibiting the anti-apoptotic BCL2 family members.¹⁸ To this end, small-molecule inhibitors, so-called BH3 mimetics, have been developed. ABT-737 and the highly related ABT-263 both inhibit BCL2, BCLXL and BCLW^{19, 20, 21} and were shown to be effective in killing cancer cells in vitro and in vivo²¹ with a preference for BCL2.^{19, 22} As BCL2 protein expression is often upregulated in hematopoietic cancers, it represents a promising target, which was supported by high efficacy of these BH3 mimetics in animal experiments.²¹ However, in vivo efficacy is limited due to thrombocytopenia, which relates to a dependence of platelets on BCLXL for survival.^{23, 24} To overcome this toxicity, a BCL2-specific compound, ABT-199, was developed.²⁵ Souers et al.²⁵ showed that inhibition of BCL2 using ABT-199 blocks tumor growth of acute lymphoblastic leukemia cells in xenografts. In addition to the single compound effects of ABT-199, combination with rituximab inhibited growth of non-Hodgkin''s lymphoma, mantle cell lymphoma and acute lymphoblastic leukemia tumor cells growth in vivo.²⁵ Moreover, highly effective tumor lysis was observed in all three patients with chronic lymphocytic leukemia that were treated with ABT-199.²⁵ More recently, a BCLXL-specific compound, WEHI-539, was discovered using high-throughput chemical screening.²⁶As the apoptotic balance appears a useful target for the treatment of cancers and CSCs have been suggested to resist therapy selectively, we set out to analyze whether specifically colon CSCs are resistant to therapy and whether this is due to an enhanced anti-apoptotic threshold, specific to CSCs. To study chemosensitivity, we developed a robust single cell-based analysis in which we can measure apoptosis simultaneously in CSCs and their differentiated progeny. Utilizing this system we showed that colon CSCs and not their differentiated progeny are resistant to chemotherapeutic compounds and that this was due to the fact that these cells are less primed to mitochondrial death. Furthermore, inhibition of anti-apoptotic BCLXL molecule with either ABT-737 or WEHI-539, but not ABT-199, breaks this resistance and sensitizes the CSCs to chemotherapy.     

Mir-660 is downregulated in lung cancer patients and its replacement inhibits lung tumorigenesis by targeting MDM2-p53 interaction

Lung cancer represents the leading cause of cancer-related death in developed countries. Despite the advances in diagnostic and therapeutic techniques, the 5-year survival rate remains low. The research for novel therapies directed to biological targets has modified the therapeutic approach, but the frequent engagement of resistance mechanisms and the substantial costs, limit the ability to reduce lung cancer mortality. MicroRNAs (miRNAs) are small noncoding RNAs with known regulatory functions in cancer initiation and progression. In this study we found that mir-660 expression is downregulated in lung tumors compared with adjacent normal tissues and in plasma samples of lung cancer patients with poor prognosis, suggesting a potential functional role of this miRNA in lung tumorigenesis. Transient and stable overexpression of mir-660 using miRNA mimics reduced migration, invasion, and proliferation properties and increased apoptosis in p53 wild-type lung cancer cells (NCI-H460, LT73, and A549). Furthermore, stable overexpression using lentiviral vectors in NCI-H460 and A549 cells inhibited tumor xenograft growth in immunodeficient mice (95 and 50% reduction compared with control, respectively), whereas the effects of mir-660 overexpression were absent in H1299, a lung cancer cell line lacking p53 locus, both in in vitro and in vivo assays. We identified and validated mouse double minute 2 (MDM2) gene, a key regulator of the expression and function of p53, as a new direct target of mir-660. In addition, mir-660 expression reduced both mRNA and protein expression of MDM2 in all cell lines and stabilized p53 protein levels resulting in an upregulation of p21^WAF1/CIP1 in p53 wild-type cells. Our finding supports that mir-660 acts as a tumor suppressor miRNA and we suggest the replacement of mir-660 as a new therapeutic approach for p53 wild-type lung cancer treatment.Lung cancer is the leading cause of cancer death worldwide, resulting in >1.4 million deaths/year.¹ Lung tumors are often discovered as locally advanced or metastatic disease, and despite improvements in molecular diagnosis and targeted therapies, the overall 5-year survival rate remains in the 10–20% range. Indeed, nonsmall cell lung cancer (NSCLC) is poorly chemosensitive to most of the available agents with response rates ranging from 10 to 25%.² The discovery of recurrent mutations in the epidermal growth factor receptor (EGFR) kinase,³ as well as gene fusion products involving the anaplastic lymphoma kinase (ALK),⁴ has led to a marked change in the treatment of patients with lung adenocarcinoma, the most common type of lung cancer.^{5, 6} To date, patients with mutations in the EGFR gene, suitable for targeting by EGFR tyrosine kinase inhibitors, represent roughly 10%, whereas the subgroup of tumors with ALK rearrangements, targeted by ALK inhibitors, is only ~5%.⁷ Thus, the majority of lung tumors lack effective treatment and novel therapeutic strategies are still needed.MicroRNAs (miRNAs) are short noncoding RNAs, 20–24 nucleotides long, that have important roles in almost all biological pathways,^{8, 9, 10, 11} and influence cancer-relevant processes, such as proliferation,¹² cell cycle,¹³ apoptosis,¹⁴ and migration.¹⁵ Many studies have reported the critical role of miRNAs in lung cancer pathogenesis and their potential as biomarkers for lung cancer risk stratification,¹⁶ outcome prediction,¹⁷ and classification of histological subtypes.^{18, 19} miRNAs are actively released by various cell types and can be detected in biological fluids, such as plasma and serum, making them suitable as circulating biomarkers in NSCLC.^{20, 21}There is limited evidence of mir-660 deregulation in cancer and little is known about its role in lung tumorigenesis and its putative target genes. Mir-660 has been reported to be upregulated in chronic lymphocytic leukemia^{22, 23} and in leukemic cells after treatment with 4-hydroxynonenal, a compound that induces differentiation and blocks proliferation of leukemic cells.²⁴ In a previous study we demonstrated that mir-660 was one of the 24 miRNAs deregulated in plasma samples of NSCLC patients identified in a low-dose computed tomography (LDCT) screening trial.²⁰The p53 tumor suppressor protein is a key regulator of cell cycle G0/G1 checkpoint, senescence, and apoptosis in response to cellular stress signals.^{25, 26} Mouse double minute 2 (MDM2), a p53–E3 ubiquitin ligase,²⁷ is the principal negative regulator of the expression level and function of p53.^{28, 29} Several studies have illustrated different mechanisms of p53 regulation by MDM2,^{30, 31} such as binding transactivation region of p53,^{32, 33} promoting nuclear export and cytoplasmic accumulation of p53 by monoubiquitination,^{34, 35} and inducing p53 proteosomal degradation by polyubiquitination.³⁶ In addition, MDM2 gene has been reported to be amplified or overexpressed in a variety of human cancers, such as sarcoma,³⁷ lymphoma,³⁸ breast cancer,³⁹ lung cancer,⁴⁰ and testicular germ cell tumor.⁴¹ Several miRNAs targeting MDM2 have been identified, such as the mir-143/mir-145 cluster that can be induced by p53,⁴² as well as mir-25 and mir-32, known to inhibit tumor glioblastoma growth in mouse brain.⁴³In this study, we report that mir-660 is downregulated in tissue and plasma samples of lung cancer patients and demonstrate that mir-660 replacement impairs the functionality of p53 wild-type (wt) lung cancer cells and inhibits in vitro and in vivo tumor growth. We showed that all the effects observed after mir-660 overexpression were absent in p53 ko cells, identified MDM2 as mir-660 direct target gene and indicate impairment of the MDM2/p53 interaction as the mechanism underlying tumor growth inhibition.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

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.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.