Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters.

The classical methods for determining glucose uptake rates in living cells involve the use of isotopically labeled 2-deoxy-d-glucose or 3-O-methyl-d-glucose, which enter cells via well-characterized membrane transporters of the SLC2A and SLC5A families, respectively. These classical methods, however, are increasingly being displaced by high-throughput assays that utilize fluorescent analogs of glucose. Among the most commonly used of these analogs are 2-NBDG and 6-NBDG, which contain a bulky 7-nitro-2,1,3-benzoxadiazol-4-yl-amino moiety in place of a hydroxy group on d-glucose. This fluorescent group significantly alters both the size and shape of these molecules compared to glucose, calling into question whether they actually enter cells by the same transport mechanisms. In this study, we took advantage of the well-defined glucose uptake mechanism of L929 murine fibroblasts, which rely exclusively on the Glut1/Slc2a1 membrane transporter. We demonstrate that neither pharmacologic inhibition of Glut1 nor genetic manipulation of its expression has a significant impact on the binding or uptake of 2-NBDG or 6-NBDG by L929 cells, though both approaches significantly impact [3H]-2-deoxyglucose uptake rates. Together these data indicate that 2-NBDG and 6-NBDG can bind and enter mammalian cells by transporter-independent mechanisms, which calls into question their utility as an accurate proxy for glucose transport.

Three-dimensional coculture provides an improved in vitro model for papillary renal cell carcinoma.

Papillary renal cell carcinoma (pRCC) represents the second most common kidney cancer and can be distinguished from other types based on its unique histological architecture and specific pattern of genomic alterations. Sporadic type 1 pRCC is almost universally driven by focal or chromosomal amplification of the receptor tyrosine kinase MET, although the specific mode of its activation is unclear. Although the MET receptors found in human tumor specimens appear highly active, those found on the surface of in vitro-cultured tumor cells are only weakly activated in the absence of exogenous hepatocyte growth factor ligand. Furthermore, pRCC cells cultured in standard two-dimensional conditions with serum fail to respond functionally to MET knockdown or the selective MET inhibitor capmatinib despite clear evidence of kinase inhibition at the molecular level. To better model pRCC in vitro, we developed a three-dimensional coculture system in which renal tumor cells are layered on top of primary fibroblasts in a fashion that mimics the papillary architecture of human tumors. In this three-dimensional spheroid model, the tumor cells survive and proliferate in the absence of serum due to trophic support of hepatocyte growth factor-producing fibroblasts. Unlike tumor cells grown in monoculture, the proliferation of cocultured tumor cells is sensitive to capmatinib and parallels inhibition of MET kinase activity. These findings demonstrate the importance of stromal fibroblasts in pRCC and indicate that accurate in vitro representation of this disease requires the presence of both tumor and fibroblast cells in a structured coculture model.NEW & NOTEWORTHY Two-dimensional monoculture of papillary renal cancer cells fails to replicate several features of the disease found in humans. We hypothesized that this discordance results from lack of trophic support from renal fibroblasts, which are involved in the architecture of human papillary renal tumors. We found that three-dimensional layering of renal cancer cells on top of a fibroblast core using magnetic bioprinting produces a structured spheroid that more faithfully mimics the behavior of human tumors.

NMS-873 functions as a dual inhibitor of mitochondrial oxidative phosphorylation.

Small-molecule inhibitors of enzyme function are critical tools for the study of cell biological processes and for treatment of human disease. Identifying inhibitors with suitable specificity and selectivity for single enzymes, however, remains a challenge. In this study we describe our serendipitous discovery that NMS-873, a compound that was previously identified as a highly selective allosteric inhibitor of the ATPase valosin-containing protein (VCP/p97), rapidly induces aerobic fermentation in cultured human and mouse cells. Our further investigation uncovered an unexpected off-target effect of NMS-873 on mitochondrial oxidative phosphorylation, specifically as a dual inhibitor of Complex I and ATP synthase. This work points to the need for caution regarding the interpretation of cell survival data associated with NMS-873 treatment and indicates that cellular toxicity associated with its use may be caused by both VCP/p97-dependent and VCP/p97-independent mechanisms.

Pharmacologic inhibition of N-linked glycan trimming with kifunensine disrupts GLUT1 trafficking and glucose uptake.

The facilitative glucose transport GLUT1 (SLC2A1) is a constitutively expressed membrane protein involved in basal uptake of blood glucose. GLUT1 modification by N-linked glycosylation at a single asparagine residue (N45) appears to play multiple roles in the trafficking, stability and transport activity of this protein. Here we examine the role of complex N-glycosylation on GLUT1 function in renal epithelial cells by arresting this modification at the high-mannose stage with the mannosidase I inhibitor kifunensine. Consistent with prior work in which GLUT1 glycosylation was completely inhibited, we find that kifunensine treatment results in a time-dependent decrease of up to 40% in cellular glucose uptake. We further demonstrate that this effect is primarily a result of deficient GLUT1 trafficking to the cell membrane due to quality control mechanisms that instead direct GLUT1 to the ER-associated degradation (ERAD) pathway. Unlike tunicamycin, which inhibits the first step in N-glycosyl transfer and causes dramatic cell cycle arrest, kifunensine causes only a modest decrease in GLUT1 levels and cell cycle progression in both normal and transformed renal cells. The effect of kifunensine on the cell cycle appears to be independent of its effect on GLUT1, since all renal cell types in this study displayed decreased proliferation regardless of their dependence on glucose uptake for growth and survival. Together these results indicate that proper N-glycan processing plays an important role in directing GLUT1 to the cell surface and that disruption of mannosidase activity results in aberrant degradation of GLUT1 by the ERAD pathway.

GLUT1 is associated with sphingolipid-organized, cholesterol-independent domains in L929 mouse fibroblast cells.

Glucose is a preferred metabolite in most mammalian cells, and proper regulation of uptake is critical for organism homeostasis. The glucose transporter 1 (GLUT1) is responsible for glucose uptake in a wide variety of cells and appears to be regulated in a tissue specific manner. Therefore, a better understanding of GLUT1 regulation within its various cellular environments is essential for developing therapeutic strategies to treat disorders associated with glucose homeostasis. Previous findings suggest that plasma membrane subdomains called lipid rafts may play a role in regulation of GLUT1 uptake activity. While studying this phenomenon in L929 mouse fibroblast cells, we observed that GLUT1 associates with a low density lipid microdomain distinct from traditionally-defined lipid rafts. These structures are not altered by cholesterol removal with methyl-ß-cyclodextrin and lack resistance to cold Triton X-100 extraction. Our data indicate that the GLUT1-containing membrane microdomains in L929 cells, as well as GLUT1's basal activity, are instead sphingolipid-dependent, being sensitive to both myriocin and sphingomyelinase treatment. These microdomains appear to be organized primarily by their lipid composition, as disruption of the actin cytoskeleton or microtubules does not alter the association of GLUT1 with them. Furthermore, the association of GLUT1 with these microdomains appears not to require palmitoylation or glycosylation, as pharmacologic inhibition of these processes had no impact on GLUT1 density in membrane fractions. Importantly, we find no evidence that GLUT1 is actively translocated into or out of low density membrane fractions in response to acute activation in L929 cell.

LRRK2 deficiency impairs trans-Golgi to lysosome trafficking and endocytic cargo degradation in human renal proximal tubule epithelial cells.

Defects in vesicular trafficking underlie a wide variety of human diseases. Genetic disruption of leucine-rich repeat kinase 2 (LRRK2) in rodents results in epithelial vesicular trafficking errors that can also be induced by treatment of animals with LRRK2 kinase inhibitors. Here we demonstrate that defects in human renal cells lacking LRRK2 phenocopy those seen in the kidneys of Lrrk2 knockout mice, characterized by accumulation of intracellular waste vesicles and fragmentation of the Golgi apparatus. This phenotype can be recapitulated by knockdown of N-ethylmaleimide-sensitive factor, which physically associates with LRRK2 in renal cells. Deficiency in either protein leads to a defect in trans-Golgi to lysosome protein trafficking, which compromises the capacity of lysosomes to degrade endocytic and autophagic cargo. In contrast, neither bulk endocytosis nor autophagic flux are impaired when LRRK2 is acutely knocked down in normal immortalized human kidney (HK2) cells. These data collectively suggest that the primary renal defect caused by LRRK2 deficiency is in protein trafficking between the Golgi apparatus and late endosome/lysosome, which leads to progressive impairments in lysosomal function.

Quercetin inhibits glucose transport by binding to an exofacial site on GLUT1.

Quercetin, a common dietary flavone, is a competitive inhibitor of glucose uptake and is also thought to be transported into cells by GLUT1. In this study, we confirm that quercetin is a competitive inhibitor of GLUT1 and also demonstrate that newly synthesized compounds, WZB-117 and BAY-876 are robust inhibitors of GLUT1 in L929 cells. To measure quercetin interaction with L929 cells, we develop a new fluorescent assay using flow cytometry. The binding of quercetin and its inhibitory effects on 2-deoxyglucose (2DG) uptake showed nearly identical dose dependent effects, with both having maximum effects between 50 and 100 µM and similar half maximum effects at 8.9 and 8.5 µM respectively. The interaction of quercetin was rapid with t1/2 of 54 s and the onset and loss of its inhibitory effects on 2DG uptake were equally fast. This suggests that either quercetin is simply binding to surface GLUT1 or its transport in and out of the cell reaches equilibrium very quickly. If quercetin is transported, the co-incubation of quercetin with other glucose inhibitors should block quercetin uptake. However, we observed that WZB-117, an exofacial binding inhibitor of GLUT1 reduced quercetin interaction, while cytochalasin B, an endofacial binding inhibitor, enhanced quercetin interaction, and BAY-876 had no effect on quercetin interaction. Taken together, these data are more consistent with quercetin simply binding to GLUT1, but not actually being transported into L929 cells via the glucose channel in GLUT1.

Caffeine inhibition of GLUT1 is dependent on the activation state of the transporter.

Caffeine has been shown to be a robust uncompetitive inhibitor of glucose uptake in erythrocytes. It preferentially binds to the nucleotide-binding site on GLUT1 in its tetrameric form and mimics the inhibitory action of ATP. Here we demonstrate that caffeine is also a dose-dependent, uncompetitive inhibitor of 2-deoxyglucose (2DG) uptake in L929 fibroblasts. The inhibitory effect on 2DG uptake in these cells was reversible with a rapid onset and was additive to the competitive inhibitory effects of glucose itself, confirming that caffeine does not interfere with glucose binding. We also report for the first time that caffeine inhibition was additive to inhibition by curcumin, suggesting distinct binding sites for curcumin and caffeine. In contrast, caffeine inhibition was not additive to that of cytochalasin B, consistent with previous data that reported that these two inhibitors have overlapping binding sites. More importantly, we show that the magnitude of maximal caffeine inhibition in L929 cells is much lower than in erythrocytes (35% compared to 90%). Two epithelial cell lines, HCLE and HK2, have both higher concentrations of GLUT1 and increased basal 2DG uptake (3-4 fold) compared to L929 cells, and subsequently display greater maximal inhibition by caffeine (66-70%). Interestingly, activation of 2DG uptake (3-fold) in L929 cells by glucose deprivation shifted the responsiveness of these cells to caffeine inhibition (35%-70%) without a change in total GLUT1 concentration. These data indicate that the inhibition of caffeine is dependent on the activity state of GLUT1, not merely on the concentration.

A mitochondrial RNAi screen defines cellular bioenergetic determinants and identifies an adenylate kinase as a key regulator of ATP levels.

Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels. We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC) perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity. Additionally, we identified a mitochondrial adenylate kinase (AK4) that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome.

Cytokine receptor-like factor 1 (CRLF1) protects against 6-hydroxydopamine toxicity independent of the gp130/JAK signaling pathway.

Oxidative stress is an important cause of cellular toxicity in the central nervous system and contributes to the pathology associated with neurodegenerative disorders including Parkinson's disease. As such, elucidation of cellular mechanisms that enhance neuronal resistance to oxidative stress may provide new avenues for therapy. In this study we employed a simple two-state cellular model to identify genes that are associated with resistance to oxidative stress induced by 6-hydroxydopamine (6-OHDA). In this model, undifferentiated neuroblastoma cells display higher sensitivity to 6-OHDA than differentiated cells. By comparing the gene expression between these two states, we identified several genes whose expression is altered concomitant with changes in 6-OHDA sensitivity. This gene set includes cytokine receptor-like factor 1 (CRLF1), which is up-regulated during the differentiation process and has been previously implicated in neuroprotection. We show that the product of this gene is both necessary and sufficient for increased resistance to 6-OHDA in differentiated neuroblastoma cells, and that CRLF1 serves its protective role by a cell autonomous mechanism that is independent from its known role as a co-ligand for the ciliary neurotrophic factor receptor. These data provide an additional role for CRLF1 that could potentially explain its broad expression pattern and effects on cells lacking expression of this receptor.

Characterization of differential protein tethering at the plasma membrane in response to epidermal growth factor signaling.

Physical tethering of membrane proteins to the cortical actin cytoskeleton provides functional organization to the plasma membrane and contributes to diverse cellular processes including cell signaling, vesicular trafficking, endocytosis, and migration. For these processes to occur, membrane protein tethering must be dynamically regulated in response to environmental cues. In this study, we describe a novel biochemical scheme for isolating the complement of plasma membrane proteins that are physically tethered to the actin cytoskeleton. We utilized this method in combination with tandem liquid chromatography/mass spectrometry (LC-MS/MS) to demonstrate that cytoskeletal tethering of membrane proteins is acutely regulated by epidermal growth factor (EGF) in normal human kidney (HK2) cells. Our results indicate that several proteins known to be involved in EGF signaling, as well as other proteins not traditionally associated with this pathway, are tethered to the cytoskeleton in dynamic fashion. Further analysis of one hit from our proteomic survey, the receptor phosphotyrosine phosphatase PTPRS, revealed a correlation between cytoskeletal tethering and endosomal trafficking in response to EGF. This finding parallels previous indications that PTPRS is involved in the desensitization of EGFR and provides a potential mechanism to coordinate localization of these two membrane proteins in the same compartment upon EGFR activation.

STAT3 is activated by JAK2 independent of key oncogenic driver mutations in non-small cell lung carcinoma.

Constitutive activation of STAT3 is a common feature in many solid tumors including non-small cell lung carcinoma (NSCLC). While activation of STAT3 is commonly achieved by somatic mutations to JAK2 in hematologic malignancies, similar mutations are not often found in solid tumors. Previous work has instead suggested that STAT3 activation in solid tumors is more commonly induced by hyperactive growth factor receptors or autocrine cytokine signaling. The interplay between STAT3 activation and other well-characterized oncogenic "driver" mutations in NSCLC has not been fully characterized, though constitutive STAT3 activation has been proposed to play an important role in resistance to various small-molecule therapies that target these oncogenes. In this study we demonstrate that STAT3 is constitutively activated in human NSCLC samples and in a variety of NSCLC lines independent of activating KRAS or tyrosine kinase mutations. We further show that genetic or pharmacologic inhibition of the gp130/JAK2 signaling pathway disrupts activation of STAT3. Interestingly, treatment of NSCLC cells with the JAK1/2 inhibitor ruxolitinib has no effect on cell proliferation and viability in two-dimensional culture, but inhibits growth in soft agar and xenograft assays. These data demonstrate that JAK2/STAT3 signaling operates independent of known driver mutations in NSCLC and plays critical roles in tumor cell behavior that may not be effectively inhibited by drugs that selectively target these driver mutations.

Combined gene expression profiling and RNAi screening in clear cell renal cell carcinoma identify PLK1 and other therapeutic kinase targets.

In recent years, several molecularly targeted therapies have been approved for clear cell renal cell carcinoma (ccRCC), a highly aggressive cancer. Although these therapies significantly extend overall survival, nearly all patients with advanced ccRCC eventually succumb to the disease. To identify other molecular targets, we profiled gene expression in 90 ccRCC patient specimens for which tumor grade information was available. Gene set enrichment analysis indicated that cell-cycle-related genes, in particular, Polo-like kinase 1 (PLK1), were associated with disease aggressiveness. We also carried out RNAi screening to identify kinases and phosphatases that when inhibited could prevent cell proliferation. As expected, RNAi-mediated knockdown of PLK1 and other cell-cycle kinases was sufficient to suppress ccRCC cell proliferation. The association of PLK1 in both disease aggression and in vitro growth prompted us to examine the effects of a small-molecule inhibitor of PLK1, BI 2536, in ccRCC cell lines. BI 2536 inhibited the proliferation of ccRCC cell lines at concentrations required to inhibit PLK1 kinase activity, and sustained inhibition of PLK1 by BI 2536 led to dramatic regression of ccRCC xenograft tumors in vivo. Taken together, these findings highlight PLK1 as a rational therapeutic target for ccRCC.

Tailoring tyrosine kinase inhibitors to fit the lung cancer genome.

Tyrosine kinase inhibitors (TKIs) have been in use as cancer therapeutics for nearly a decade, and their utility in targeting specific malignancies with defined genetic lesions has proven to be remarkably effective. Recent efforts to characterize the spectrum of genetic lesions found in non-small cell lung carcinoma (NSCLC) have provided important insights into the molecular basis of this disease and have also revealed a wide array of tyrosine kinases that might be effectively targeted for rationally designed therapies. The findings of these studies, however, also provide a cautionary tale about the limitations of single-agent therapies, which fail to account for the genetic heterogeneity and pathway redundancy that characterize advanced NSCLC. Emergence of drug resistance mechanisms to specific TKIs, such as gefitinib and erlotinib, suggests that more sophisticated chemotherapeutic paradigms that target multiple pathways at the same time will be required to effectively treat this disease.

Identification of PTPsigma as an autophagic phosphatase.

Macroautophagy is a dynamic process whereby portions of the cytosol are encapsulated in double-membrane vesicles and delivered to the lysosome for degradation. Phosphatidylinositol-3-phosphate (PtdIns3P) is concentrated on autophagic vesicles and recruits effector proteins that are crucial for this process. The production of PtdIns3P by the class III phosphatidylinositol 3-kinase Vps34, has been well established; however, protein phosphatases that antagonize this early step in autophagy remain to be identified. To identify such enzymes, we screened human phosphatase genes by RNA interference and found that loss of PTPσ, a dual-domain protein tyrosine phosphatase (PTP), increases levels of cellular PtdIns3P. The abundant PtdIns3P-positive vesicles conferred by loss of PTPσ strikingly phenocopied those observed in cells starved of amino acids. Accordingly, we discovered that loss of PTPσ hyperactivates both constitutive and induced autophagy. Finally, we found that PTPσ localizes to PtdIns3P-positive membranes in cells, and this vesicular localization is enhanced during autophagy. We therefore describe a novel role for PTPσ and provide insight into the regulation of autophagy. Mechanistic knowledge of this process is crucial for understanding and targeting therapies for several human diseases, including cancer and Alzheimer's disease, in which abnormal autophagy might be pathological.

Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required for oncogenic MET signaling in papillary renal and thyroid carcinomas.

The receptor tyrosine kinase MET is frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence of growth factor stimulation by its endogenous ligand, hepatocyte growth factor (HGF). We sought to identify mechanisms of signaling crosstalk that promote MET activation by searching for kinases that are coordinately dysregulated with wild-type MET in human tumors. Our bioinformatic analysis identified leucine-rich repeat kinase-2 (LRRK2), which is amplified and overexpressed in papillary renal and thyroid carcinomas. Down-regulation of LRRK2 in cultured tumor cells compromises MET activation and selectively reduces downstream MET signaling to mTOR and STAT3. Loss of these critical mitogenic pathways induces cell cycle arrest and cell death due to loss of ATP production, indicating that MET and LRRK2 cooperate to promote efficient tumor cell growth and survival in these cancers.

Inhibin-A antagonizes TGFbeta2 signaling by down-regulating cell surface expression of the TGFbeta coreceptor betaglycan.

Inhibin is an atypical member of the TGFbeta family of signaling ligands and is classically understood to function via competitive antagonism of activin ligand binding. Inhibin-null (Inha-/-) mice develop both gonadal and adrenocortical tumors, the latter of which depend upon gonadectomy for initiation. We have previously shown that gonadectomy initiates adrenal tumorigenesis in Inha-/- mice by elevating production of LH, which drives aberrant proliferation and differentiation of subcapsular adrenocortical progenitor cells. In this study, we demonstrate that LH signaling specifically up-regulates expression of TGFbeta2 in the subcapsular region of the adrenal cortex, which coincides with regions of aberrant Smad3 activation in Inha-/- adrenal glands. Consistent with a functional interaction between inhibin and TGFbeta2, we further demonstrate that recombinant inhibin-A antagonizes signaling by TGFbeta2 in cultured adrenocortical cells. The mechanism of this antagonism depends upon the mutual affinity of inhibin-A and TGFbeta2 for the signaling coreceptor betaglycan. Although inhibin-A cannot physically displace TGFbeta2 from its binding sites on betaglycan, binding of inhibin-A to the cell surface causes endocytic internalization of betaglycan, thereby reducing the number of available binding sites for TGFbeta2 on the cell surface. The mechanism by which inhibin-A induces betaglycan internalization is clathrin independent, making it distinct from the mechanism by which TGFbeta ligands themselves induce betaglycan internalization. These data indicate that inhibin can specifically antagonize TGFbeta2 signaling in cellular contexts where surface expression of betaglycan is limiting and provide a novel mechanism for activin-independent phenotypes in Inha-/- mice.

Genetic removal of Smad3 from inhibin-null mice attenuates tumor progression by uncoupling extracellular mitogenic signals from the cell cycle machinery.

Inhibin and activin are members of the TGFbeta family that perform mutually antagonistic signaling roles in the anterior pituitary, gonads, and adrenal gland. Unopposed activin signaling in inhibin-null (Inha-/-) mice causes the formation of granulosa cell tumors in the gonads and adrenal cortex, which depend upon FSH for efficient growth and progression. In this study, we demonstrate that Smad3, a key effector of activin signaling, is expressed at high levels and is constitutively activated in tumors from these mice. Removal of Smad3 from Inha-/- mice by a genetic cross to Smad3-null (Madh3-/-) mice leads to a significant decrease in cyclinD2 expression and a significant attenuation of tumor progression in the gonads and adrenal. The decrease in cyclinD2 levels in compound knockout mice is related to a reduction in mitogenic signaling through the phosphoinositide-3-kinase (PI3-kinase)/Akt pathway, which is required for normal cell cycle progression in tumor cells. Loss of PI3-kinase/Akt signaling cannot be attributed to alterations in IGF expression, suggesting instead that signaling through the FSH receptor is attenuated. Gene expression profiling in the ovaries of Madh3-/- and Inha-/-:Madh3-/- compound knockout mice supports this hypothesis and further suggests that Smad3 is specifically required for FSH to activate PI3-kinase/Akt, but not protein kinase A. Together these observations imply that activin/Smad3 signaling is necessary for efficient signaling by FSH in Inha-/- tumor cells and that interruption of this pathway uncouples FSH from its intracellular mitogenic effectors.

Origin and identity of adrenocortical tumors in inhibin knockout mice: implications for cellular plasticity in the adrenal cortex.

Inhibin knockout (Inha-/-) mice develop gonadal sex-cord tumors and--when gonadectomized--adrenocortical tumors. Previous reports demonstrated that adrenocortical tumors from Inha-/- mice produce estrogen and depend on gonadotropin signaling for initiation. Here we show that, in addition to producing estrogen, the adrenocortical tumors display a global change in cellular identity, composed of two unique cell types expressing differing arrays of genes normally restricted to theca and granulosa cells of the ovary. Many of these genes are also induced in wild-type adrenals after gonadectomy or upon chronic gonadotropin stimulation, suggesting that the adrenal cortex normally contains a population of pluripotent cells that can be driven toward an adrenal or gonadal identity given the appropriate pituitary stimuli. A central feature of this altered cellular identity is the switch from predominant expression of Gata6 (endogenous to the adrenal cortex) to Gata4, which defines cellular identity in the ovary. We show that stable transfection of Gata4 in cultured adrenocortical cells is sufficient to activate ovarian-specific genes of both theca and granulose lineages. Spatial analysis of Gata4 expression reveals a distinct pattern of localization to the supcapsular region of the adrenal, which contains undifferentiated progenitor cells that continuously populate the adrenocortical zones. Although both wild-type and Inha-/- mice display this pattern, only Inha-/- mice produce tumors composed of these Gata4-positive cells. These data suggest that Inha-/- adrenocortical tumors cells are derived from pluripotent adrenocortical progenitor cells that adopt a gonadal fate due to the convergent loss of inhibin and chronic exposure to elevated gonadotropins.