An antisense oligonucleotide targeting TGF-ß2 inhibits lung metastasis and induces CD86 expression in tumor-associated macrophages.

BACKGROUND: The transforming growth factor (TGF)-ß pathway is a well-described inducer of immunosuppression and can act as an oncogenic factor in advanced tumors. Several preclinical and clinical studies show that the TGF-ß pathway can be considered a promising molecular target for cancer therapy. The human genome has three TGF-ß isoforms and not much is known about the oncogenic response to each of the isoforms. Here, we studied the antitumor response to ISTH0047, a recently developed locked nucleic acid-modified antisense oligonucleotide targeting TGF-ß2. MATERIALS AND METHODS: We have studied the anticancer response to ISTH0047 using gymnotic delivery in tumor cell cultures and in in vivo preclinical orthotopic mouse models for primary tumors (breast and kidney tumors) and lung metastasis. RESULTS: We observed that ISTH0047 is able to significantly reduce TGF-ß2 mRNA and protein levels without altering the levels of TGF-ß1 and TGF-ß3. ISTH0047 prevented lung metastasis in syngeneic orthotopic renal cell carcinoma (RENCA) and breast cancer (4T1) tumor models. In addition, using an orthotopic xenograft model of a lung cancer cell line (CRL5807) that mainly expresses TGF-ß2, we observed that ISTH0047 had an important effect on the lung microenvironment inhibiting the growth of lung lesions. ISTH0047 treatment re-educated macrophages in the lung parenchyma to express the tumor-suppressive factor, CD86. CONCLUSION: Overall, our data point to TGF-ß2 as a therapeutic target and ISTH0047 as a novel anticancer drug to prevent lung metastasis by impacting on the tumor niche, in part, through the induction of CD86 in tumor-associated macrophages.

Survivin drives tumor-associated macrophage reprogramming: a novel mechanism with potential impact for obesity.

PURPOSE: Recent studies point to adipose-derived stem cells (ASCs) as a link between obesity and cancer. We aimed to determine whether survivin, which is highly secreted by ASCs from subjects with obesity, might drive a pro-tumoral phenotype in macrophages. METHODS: The effect of ASC conditioned medium on the macrophage phenotype was assessed by expression studies. Survivin intracellular localization and internalization were examined by subcellular fractionation and immunofluorescence, respectively. Loss- and gain-of-function studies were performed using adenoviral vectors, and gene expression patterns, migration and invasion capacities of cancer cells were examined. Heterotypic cultures of ASCs, macrophages and cancer cells were established to mimic the tumor microenvironment. Survivin-blocking experiments were used to determine the impact of survivin on both macrophages and cancer cells. Immunohistochemical analysis of survivin was performed in macrophages from ascitic fluids of cancer patients and healthy controls. RESULTS: We found that obese-derived ASCs induced a phenotypic switch in macrophages characterized by the expression of both pro- and anti-inflammatory markers. Macrophages were found to internalize extracellular survivin, generating hybrid macrophages with a tumor-associated phenotype that included secretion of survivin. Exogenous expression of survivin in macrophages generated a similar phenotype and enhanced the malignant characteristics of cancer cells by a mechanism dependent on survivin phosphorylation at threonine 34. Survivin secreted by both ASCs from subjects with obesity and tumor-associated macrophages synergistically boosted the malignancy of cancer cells. Importantly, survivin was mainly detected in ascites-associated macrophages from patients with a malignant diagnosis. CONCLUSION: Our data indicate that survivin may serve as a molecular link between obesity and cancer and as a novel marker for tumor-associated macrophages.

Transport of nucleoside analogs across the plasma membrane: a clue to understanding drug-induced cytotoxicity.

Nucleoside analogs are widely used in the treatment of cancer and viral-induced diseases. Efficacy of treatments relies upon a variety of events, including transport across tissue and target barriers, which determine drug pharmacokinetics and target cell bioavailability. To exert their action, nucleosides have to be chemically modified, thus compromising cellular uptake by those routes which are responsible for the uptake of natural nucleosides and nucleobases. In this review we will focus on established knowledge and recent advances in the understanding of nucleoside- and nucleobase-derived drug uptake mechanisms. Basically, these drug uptake processes involve the gene families SLC22, SLC28 and SLC29. These gene families encode Organic Anion Transporter (OAT)/Organic Cation Transporter (OCT), Concentrative Nucleoside Transporter (CNT) and Equilibrative Nucleoside Transporter (ENT) proteins, respectively. The pharmacological profiles of these plasma membrane carriers as well as their basic physiological and regulatory properties, including their tissue and subcellular distribution will be reviewed. This knowledge is crucial for the understanding of nucleoside- and nucleobase-derived drug bioavailability and therapeutic action. Moreover, changes in both transporter expression and/or transporter function (for instance as a consequence of gene variability) might also modulate response to treatment, thereby anticipating a putative diagnostic and predictive added value to the analysis of transporter expression and their corresponding genetic variants.

Equilibrative nucleoside transporter-2 (hENT2) protein expression correlates with ex vivo sensitivity to fludarabine in chronic lymphocytic leukemia (CLL) cells.

Fludarabine is considered the treatment of choice for most patients with chronic lymphocytic leukemia (CLL). We have analyzed the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in CLL cells. Among the known plasma membrane transporters, we have previously observed a significant correlation between fludarabine uptake via ENT carriers and ex vivo sensitivity of CLL cells to fludarabine, although mRNA amounts of the equilibrative nucleoside transporters hENT1 and hENT2 do not show any predictive response to treatment. In this study, using polyclonal monospecific antibodies we have observed a significant correlation between the expression of hENT2 by Western blot and fludarabine uptake via hENT carriers and also with ex vivo sensitivity of CLL cells to fludarabine. These results suggest that the equilibrative nucleoside transporter hENT2 plays a role in fludarabine responsiveness in CLL patients.

Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation-independent manner.

Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology.

The concentrative nucleoside transporter family (SLC28): new roles beyond salvage?

The concentrative nucleoside transporter (CNT) family (SLC28) has three members: SLC28A1 (CNT1), SLC28A2 (CNT2) and SLC28A3 (CNT3). The CNT1 and CNT2 transporters are co-expressed in liver parenchymal cells and macrophages, two suitable models in which to study cell cycle progression. Despite initial observations suggesting that these transporter proteins might contribute to nucleoside salvage during proliferation, their subcellular localization and regulatory properties suggest alternative roles in cell physiology. In particular, CNT2 is a suitable candidate for modulation of purinergic responses, since it is under the control of the adenosine 1 receptor. Increasing evidence also suggests a role for CNT2 in energy metabolism, since its activation relies on the opening of ATP-sensitive K(+) channels. Animal and cell models genetically modified to alter nucleoside transporter expression levels may help to elucidate the particular roles of CNT proteins in cell physiology.