Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a high percentage of morbidity. The deciphering and identification of novel targets and tools for intervening with its adverse progression are therefore of immense importance. To address this goal we adopted a specific inhibitor of the intracellular tyrosine kinase Fer, whose expression level is upregulated in PDAC tumors, and is associated with poor prognosis of patients. Subjecting PDAC cells to the E260-Fer inhibitor, unraveled its simultaneous effects on the mitochondria, and on a non-mitochondrial ERK1/2 regulatory cascade. E260 caused severe mitochondrial deformation, resulting in cellular- aspartate and ATP depletion, and followed by the activation of the metabolic sensor AMPK. This led to the phosphorylation and deactivation of the bona fide AMPK substrate, RAPTOR, which serves as a positive regulator of the mTORC1 metabolic hub. Accordingly, this resulted in the inhibition of the mTORC1 activity. In parallel, E260 downregulated the activation state of the ERK1/2 kinases, and their ability to neutralize the mTORC1 suppressor TSC2, thereby accentuating the inhibition of mTORC1. Importantly, both activation of AMPK and downregulation of ERK1/2 and mTORC1 were also achieved upon the knockdown of Fer, corroborating the regulatory role of Fer in these processes. Concomitantly, in PDAC tumors and not in healthy pancreatic tissues, the expression levels of Fer demonstrate moderate but statistically significant positive correlation with the expression levels of mTOR and its downstream effector LARP1. Finally, targeting the Fer driven activation of mTORC1, culminated in necrotic death of the treated PDAC cells, envisaging a new intervention tool for the challenging PDAC disease.

Supplementary Table S2: summarizing tumor progression in immuno-compromised mice, up to 6 weeks after being subcutaneously injected with 4X106 NIH3T3-MTS- MycFerT, NIH3T3-MycFerT or NIH3T3-pIRES cells.

Supplementary Figure S1: shows the induction of cellular ROS levels upon the down-regulation of Fer or FerT using different siRNA's.

Supplementary Figure S2: demonstrates the different tumor formation promoting activities of mitochondrial directed and undirected FerT in immuno-compromised mice

Supplementary Table S1: lists the different siRNA's used in the currently described work

Supplementary Data legends. This File contains the legends for the supplementary Figures and tables

The physiological acrosome reaction occurs after mammalian spermatozoa undergo a process called capacitation in the female reproductive tract. Only acrosome reacted spermatozoon can penetrate the egg zona-pellucida and fertilize the egg. Sperm also contain several mechanisms that protect it from undergoing spontaneous acrosome reaction (sAR), a process that can occur in sperm before reaching proximity to the egg and that abrogates fertilization. We previously showed that calmodulin-kinase II (CaMKII) and phospholipase D (PLD) are involved in preventing sAR through two distinct pathways that enhance F-actin formation during capacitation. Here, we describe a novel additional pathway involving the tyrosine kinase Fer in a mechanism that also prevents sAR by enhancing actin polymerization during sperm capacitation. We further show that protein-kinase A (PKA) and the tyrosine-kinase Src, as well as PLD, direct Fer phosphorylation/activation. Activated Fer inhibits the Ser/Thr phosphatase PP1, thereby leading to CaMKII activation, actin polymerization, and sAR inhibition.

Metabolic plasticity is a hallmark of the ability of metastatic cancer cells to survive under stressful conditions. The intracellular Fer kinase is a selective constituent of the reprogramed mitochondria and metabolic system of cancer cells. In the current work, we deciphered the modulatory roles of Fer in the reprogrammed metabolic systems of metastatic, lung (H358), non-small cell lung cancer (NSCLC), and breast (MDA-MB-231), triple-negative breast cancer (TNBC), carcinoma cells. We show that H358 cells devoid of Fer (H358ΔFer), strictly depend on glucose for their proliferation and growth, and fail to compensate for glucose withdrawal by oxidizing and metabolizing glutamine. Furthermore, glucose deficiency caused increased reactive oxygen species (ROS) production and induction of a DNA damage response (DDR), accompanied by the onset of apoptosis and attenuated cell-cycle progression. Analysis of mitochondrial function revealed impaired respiratory and electron transport chain (ETC) complex 1 (comp. I) activity in the Fer-deficient H358ΔFer cells. This was manifested by decreased levels of NAD+ and ATP and relatively low abundance of tricarboxylic acid (TCA) cycle metabolites. Impaired electron transport chain comp. I activity and dependence on glucose were also confirmed in Fer-deficient, MDA-MB-231ΔFer cells. Although both H358ΔFer and MDA-MB-231ΔFer cells showed a decreased aspartate level, this seemed to be compensated by the predominance of pyrimidines synthesis over the urea cycle progression. Notably, absence of Fer significantly impeded the growth of H358ΔFer and MDA-MB-231ΔFer xenografts in mice provided with a carb-deficient, ketogenic diet. Thus, Fer plays a key role in the sustention of metabolic plasticity of malignant cells. In compliance with this notion, targeting Fer attenuates the progression of H358 and MDA-MB-231 tumors, an effect that is potentiated by a glucose-restrictive diet.

Insulin-regulated glucose homeostasis is a critical and intricate physiological process, of which not all regulatory components have been deciphered. One of the key players in modulating glucose uptake by cells is the glucose transporter-GLUT4. In this study, we aimed to explore the regulatory role of the trans-Golgi-associated protein-TATA Element Modulatory Factor (TMF1) in the GLUT4 mediated, insulin-directed glucose uptake. By establishing and using TMF1-/- myoblasts and mice, we examined the effect of TMF1 absence on the insulin driven functioning of GLUT4. We show that TMF1 is upregulated by insulin in myoblasts, and is essential for the formation of insulin responsive, glucose transporter GLUT4-containing vesicles. Absence of TMF1 leads to the retention of GLUT4 in perinuclear compartments, and to severe impairment of insulin-stimulated GLUT4 trafficking throughout the cytoplasm and to the cell plasma membrane. Accordingly, glucose uptake is impaired in TMF1-/- cells, and TMF1-/- mice are hyperglycemic. This is reflected by the mice impaired blood glucose clearance and increased blood glucose level. Correspondingly, TMF1-/- animals are leaner than their normal littermates. Thus, TMF1 is a novel effector of insulin-regulated glucose homeostasis, and dys-functioning of this protein may contribute to the onset of a diabetes-like disorder.

Aerobic glycolysis is an important metabolic adaptation of cancer cells. However, there is growing evidence that reprogrammed mitochondria also play an important metabolic role in metastatic dissemination. Two constituents of the reprogrammed mitochondria of cancer cells are the intracellular tyrosine kinase Fer and its cancer- and sperm-specific variant, FerT. Here, we show that Fer and FerT control mitochondrial susceptibility to therapeutic and hypoxic stress in metastatic colon (SW620) and non-small cell lung cancer (NSCLC-H1299) cells. Fer- and FerT-deficient SW620 and H1299 cells (SW∆Fer/FerT and H∆Fer/FerT cells, respectively) become highly sensitive to metformin treatment and to hypoxia under glucose-restrictive conditions. Metformin impaired mitochondrial functioning that was accompanied by ATP deficiency and robust death in SW∆Fer/FerT and H∆Fer/FerT cells compared to the parental SW620 and H1299 cells. Notably, selective knockout of the fer gene without affecting FerT expression reduced sensitivity to metformin and hypoxia seen in SW∆Fer/FerT cells. Thus, Fer and FerT modulate the mitochondrial susceptibility of metastatic cancer cells to hypoxia and metformin. Targeting Fer/FerT may therefore provide a novel anticancer treatment by efficient, selective, and more versatile disruption of mitochondrial function in malignant cells.

Disruption of the reprogrammed energy management system of malignant cells is a prioritized goal of targeted cancer therapy. Two regulators of this system are the Fer kinase, and its cancer cell specific variant, FerT, both residing in subcellular compartments including the mitochondrial electron transport chain. Here, we show that a newly developed inhibitor of Fer and FerT, E260, selectively evokes metabolic stress in cancer cells by imposing mitochondrial dysfunction and deformation, and onset of energy-consuming autophagy which decreases the cellular ATP level. Notably, Fer was also found to associate with PARP-1 and E260 disrupted this association thereby leading to PARP-1 activation. The cooperative intervention with these metabolic pathways leads to energy crisis and necrotic death in malignant, but not in normal human cells, and to the suppression of tumors growth in vivo. Thus, E260 is a new anti-cancer agent which imposes metabolic stress and cellular death in cancer cells.The tyrosine-kinases Fer/FerT associate with the mitochondrial electron transport chain in cancer cells supporting their metabolic reprogramming. Here the authors discover a compound that disrupts Fer /FerT activity and selectively induces cell death of cancer cell lines displaying anti-tumor activity in vivo.

TMF/ARA160 is known to be a TATA element Modulatory Factor (TMF). It was initially identified as a DNA-binding factor and a coactivator of the Androgen receptor. It was also characterized as a Golgi-associated protein, which is essential for acrosome formation during functional sperm development. However, the molecular roles of TMF in this intricate process have not been revealed. Here, we show that during spermiogenesis, TMF undergoes a dynamic change of localization throughout the Golgi apparatus. Specifically, TMF translocates from the cis-Golgi to the trans-Golgi network and to the emerging vesicles surface, as the round spermatids develop. Notably, lack of TMF led to an abnormal spatial orientation of the Golgi and to the deviation of the trans-Golgi surface away from the nucleus of the developing round spermatids. Concomitantly, pro-acrosomal vesicles derived from the TMF-/- Golgi lacked targeting properties and did not tether to the spermatid nuclear membrane thereby failing to form the acrosome anchoring scaffold, the acroplaxome, around the cell-nucleus. Absence of TMF also perturbed the positioning of microtubules, which normally lie in proximity to the Golgi and are important for maintaining Golgi spatial orientation and dynamics and for chromatoid body formation, which is impaired in TMF-/- spermatids. In-silico evaluation combined with molecular and electron microscopic analyses revealed the presence of a microtubule interacting domain (MIT) in TMF, and confirmed the association of TMF with microtubules in spermatogenic cells. Furthermore, the MIT domain in TMF, along with microtubules integrity, are required for stable association of TMF with the Golgi apparatus. Collectively, we show here for the first time that a Golgi and microtubules associated protein is crucial for maintaining proper Golgi orientation during a cell developmental process.

Tata Element Modulatory Factor (TMF/ARA160) is a multifunctional Golgi-associated protein, which accumulates in colonic enterocytes and goblet cells. Mice lacking TMF/ARA160 (TMF-/-) produce thick and uniform colonic mucus that resists adherent bacterial colonization and diminishes susceptibility of these mice to induced acute colitis, through a mechanism that is not fully understood. Here, we show that mucus secretion by goblet cells is altered in the colon of TMF-/- mice, resulting in the formation of a highly oligomerized colonic gel-forming mucin, MUC2. Microbiome analysis revealed a shift in the microbiota of TMF-/- mice leading to predominance of the Firmicutes phylum and a significantly higher abundance of probiotic beneficial bacterial species. Notably, this trait was transmissible, and when cohoused with wild-type animals, TMF-/- mice influenced the microbiota and diminished the susceptibility of wildtype mice to chemically induced dextran sulfate sodium colitis. Thus, altered mucus secretion in TMF-/- mouse colons is accompanied by a reprogrammed intestinal microbiota, leading to a transmissible reduced sensitivity to induced colitis.

Abstract The kinase Fer and its spermatogenic meiotic variant, FerT, are coexpressed in normal testes and cancerous tumors, but whether they exert related roles in spermatogenic or malignant cells has not been known. Here, we show that Fer and FerT reside in the mitochondria of spermatogenic cells and are harnessed to the reprogrammed mitochondria of colon carcinoma cells. Both kinases bound complex I of the mitochondrial electron transport chain (ETC) in spermatogenic and in colon carcinoma cells, and silencing of either Fer or FerT was sufficient to impair the activity of this complex. Directed mitochondrial accumulation of FerT in nonmalignant NIH3T3 cells increased their ETC complex I activity, ATP production, and survival, contingent upon stress conditions caused by nutrient and oxygen deprivation. Strikingly, directed mitochondrial accumulation of FerT endowed nonmalignant cells with tumor-forming ability. Thus, recruitment of a meiotic mitochondrial component to cancer cell mitochondria highlights a pivotal role for reprogrammed mitochondria in tumorigenesis. Cancer Res; 74(22); 6474–85. ©2014 AACR.

TMF/ARA160 is a Golgi-associated protein, which is essential for spermiogenesis. In this study, we show that lack of TMF/ARA160 leads to defects in both the testis and the epididymis. In the testis, spermatid retention and extensive proliferation of Leydig cells were observed. Concomitantly, the serum levels of luteinizing hormone (LH), a stimulator of Leydig cell proliferation, were significantly increased in TMF(-/-) mice. Structural and functional defects were also seen in the epididymis. These included apoptosis of epithelial epididymal cells and sperm stasis in the cauda. Notably, the serum testosterone levels of TMF(-/-) mice were significantly lower than those of wt mice, and external testosterone administration decreased the number of apoptotic epithelial epididymal cells in TMF(-/-) animals. In summary, we show here for the first time that TMF/ARA160 participates in the control of serum testosterone levels in males, and its absence results in major testicular and epididymal defects.

Fer is an intracellular tyrosine kinase that accumulates in most mammalian tissues. A truncated variant of Fer, FerT, is uniquely detected in spermatogenic cells and is absent from normal somatic tissues. Here, we show that in addition to Fer, FerT also accumulates in CC cells and in metastases derived from colorectal tumors, but not in normal human cells. Thus, FerT is a new member of the CTA protein family. Transcription of the ferT gene in CC cells was found to be driven by an intronic promoter residing in intron 10 of the fer gene and to be regulated by another CTA, the Brother of the Regulator of Imprinted Sites (BORIS) transcription factor. BORIS binds to the ferT promoter and down-regulation of BORIS significantly decreases the expression of ferT in CC cells. Accumulation of the ferT RNA was also regulated by the DNA methylation status and paralleled the expression profile of the boris transcript. Accordingly, the intronic ferT promoter was found to be hypomethylated in cancer cells expressing the FerT protein, by comparison with non-expressers. Collectively, we show here that FerT is a new CTA whose accumulation in CC cells, commonly considered low CTA expressers, is controlled by a novel transcription regulatory mechanism.

Background: TMF/ARA160 regulates the NF-κB subunit, p65 RelA, under stress conditions.Results: Loss of TMF/ARA160 renders mice colonic mucus refractory to bacterial colonization and diminishes intestinal susceptibility to acute colitis.Conclusion: TMF/ARA160 affects intestinal susceptibility to acute colitis.Significance: Targeting TMF/ARA160 in the colon may attenuate the onset and progression of acute colitis.

Fer is an intracellular tyrosine kinase which resides in both the cytoplasm and nucleus of mammalian cells. This kinase was also found in all malignant cell-lines analyzed and was shown to support cell-cycle progression in cancer cells. Herein we show that knock-down of Fer, both, impairs cell-cycle progression and imposes programmed cell death in colon carcinoma (CC) cells. The cell-cycle arrest and apoptotic death invoked by the depletion of Fer were found to depend on the activity of p53. Accordingly, down regulation of Fer led to the activation of the Ataxia Telangiectasia Mutated protein (ATM) and its down-stream effector-p53. Knock-down of Fer also increased the level of Reactive-Oxygen Species (ROS) in CC cells, and subjection of Fer depleted cells to ROS neutralizing scavengers significantly decreased the induced phosphorylation and activation of ATM and p53. Notably, over-expression of Fer opposed the Doxorubicin driven activation of ATM and p53, which can be mediated by ROS. Collectively, our findings imply that Fer sustains low ROS levels in CC cells, thereby restraining the activation of ATM and p53 in these cells.