SSRP1 Worsens Malignant Melanoma Progression by Activating MAPKs Pathway.

OBJECTIVE: Structure-specific recognition protein 1 (SSRP1) is highly expressed in a variety of tumors and promotes cell proliferation and migration. Malignant melanoma is a highly malignant skin malignancy with low morbidity and high mortality. The role of SSRP1 in malignant melanoma is still unclear. Thus, this study is intended to investigate the role of SSRP1 in malignant melanoma and reveal the related mechanisms. METHODS: Western blots and immunohistochemistry assays were used to determine the expression of SSRP1 in benign nevi tissues and malignant melanoma tissues. The si-SSRP1 was used to knockdown the expression level of SSRP1 in A375 cells. Cell proliferation was assessed by MTT assay. Wound healing and Transwell assay were performed for detected cell migratory and invasive activities, respectively. Besides, the expression levels of epithelial-mesenchymal transition (EMT) markers and MAPKs signaling pathway were measured by western blot. RESULTS: The results showed that SSRP1 was highly expressed in malignant melanoma tissues and cells, and its expression in metastatic melanoma tissues was significantly higher than that in primary melanoma. Besides, high expression level of SSRP1 was accompanied with poor prognosis in malignant melanoma patients. SSRP1 knockdown inhibited the melanoma cell proliferation, migration, and invasion. Besides, SSRP1 knockdown inhibited the process of EMT by upregulating E-cadherin, and downregulating N-cadherin and vimentin. Further studies revealed that SSRP1 silencing affected MAPK signaling pathway and reduced its phosphorylation activity in melanoma cells. CONCLUSIONS: These results suggested that SSRP1 may promote the proliferation, migration, and invasion of melanoma cells through MAPK signaling pathway. SSRP1 is closely related to the malignancy of melanoma and may be a potential target for its clinical treatment.

CD8+ T cell dysfunction by TOX intoxication: a protumorigenic event in the tumor microenvironment.

Accumulating evidence suggests the role of cellular components in achieving antitumor to protumor microenvironments. Among the various types of cells within the tumor niche, the state of CD8+ T cells apparently changes from cytotoxic T effector cells and memory T cells to exhausted CD8+ T cells. These changes in the phenotype of CD8+ T cells promote the protumor microenvironment. Recently, comprehensive experimental data delineated the role of thymocyte selection-associated high-mobility group-box protein (TOX), which regulates the transcriptional process and epigenetic remodeling, with implications in tumor and chronic viral infections. This perspective summarizes the molecular mechanisms that link CD8+ T cells, TOX, and transcriptional and epigenetic reprogramming as well as future directions for determining new avenues of cancer therapeutics.

Lay abstract Cellular components within the tumor are related to the success and failure of anticancer drugs for patients. The reasons behind the changes from antitumor to protumor microenvironments are being explored to understand the immune cells. Among several types of cells, the state of CD8⁺ cells in the immune system apparently changes from cytotoxic immune effector cells and memory effector cells to depleted CD8⁺ immune cells. These changes in the phenotype of CD8⁺ T cells promote a favorable tumor microenvironment. This minireview summarizes the importance of CD8⁺ immune cells and their regulation in the development of anticancer drugs.

Histone chaperone FACT complex inhibitor CBL0137 interferes with DNA damage repair and enhances sensitivity of medulloblastoma to chemotherapy and radiation.

Medulloblastoma (MB) is a malignant pediatric brain tumor with a poor prognosis. Post-surgical radiation and cisplatin-based chemotherapy have been a mainstay of treatment, which often leads to substantial neurocognitive impairments and morbidity, highlighting the need for a novel therapeutic target to enhance the sensitivity of MB tumors to cytotoxic therapies. We performed a comprehensive study using a cohort of 71 MB patients' samples and pediatric MB cell lines and found that MB tumors have elevated levels of nucleosome remodeling FACT (FAcilitates Chromatin Transcription) complex and DNA repair enzyme AP-endonuclease1 (APE1). FACT interacts with APE1 and facilitates recruitment and acetylation of APE1 to promote repair of radiation and cisplatin-induced DNA damage. Further, levels of FACT and acetylated APE1 both are correlate strongly with MB patients' survival. Targeting FACT complex with CBL0137 inhibits DNA repair and alters expression of a subset of genes, and significantly improves the potency of cisplatin and radiation in vitro and in MB xenograft. Notably, combination of CBL0137 and cisplatin significantly suppressed MB tumor growth in an intracranial orthotopic xenograft model. We conclude that FACT complex promotes chemo-radiation resistance in MB, and FACT inhibitor CBL0137 can be used as a chemo-radiation sensitizer to augment treatment efficacy and reduce therapy-related toxicity in high-risk pediatric patients.

The metabesity factor HMG20A potentiates astrocyte survival and reactive astrogliosis preserving neuronal integrity.

Rationale: We recently demonstrated that the 'Metabesity' factor HMG20A regulates islet beta-cell functional maturity and adaptation to physiological stress such as pregnancy and pre-diabetes. HMG20A also dictates central nervous system (CNS) development via inhibition of the LSD1-CoREST complex but its expression pattern and function in adult brain remains unknown. Herein we sought to determine whether HMG20A is expressed in the adult CNS, specifically in hypothalamic astrocytes that are key in glucose homeostasis and whether similar to islets, HMG20A potentiates astrocyte function in response to environmental cues. Methods: HMG20A expression profile was assessed by quantitative PCR (QT-PCR), Western blotting and/or immunofluorescence in: 1) the hypothalamus of mice exposed or not to either a high-fat diet or a high-fat high-sucrose regimen, 2) human blood leukocytes and adipose tissue obtained from healthy or diabetic individuals and 3) primary mouse hypothalamic astrocytes exposed to either high glucose or palmitate. RNA-seq and cell metabolic parameters were performed on astrocytes treated or not with a siHMG20A. Astrocyte-mediated neuronal survival was evaluated using conditioned media from siHMG20A-treated astrocytes. The impact of ORY1001, an inhibitor of the LSD1-CoREST complex, on HMG20A expression, reactive astrogliosis and glucose metabolism was evaluated in vitro and in vivo in high-fat high-sucrose fed mice. Results: We show that Hmg20a is predominantly expressed in hypothalamic astrocytes, the main nutrient-sensing cell type of the brain. HMG20A expression was upregulated in diet-induced obesity and glucose intolerant mice, correlating with increased transcript levels of Gfap and Il1b indicative of inflammation and reactive astrogliosis. Hmg20a transcript levels were also increased in adipose tissue of obese non-diabetic individuals as compared to obese diabetic patients. HMG20A silencing in astrocytes resulted in repression of inflammatory, cholesterol biogenesis and epithelial-to-mesenchymal transition pathways which are hallmarks of reactive astrogliosis. Accordingly, HMG20A depleted astrocytes exhibited reduced mitochondrial bioenergetics and increased susceptibility to apoptosis. Neuron viability was also hindered in HMG20A-depleted astrocyte-derived conditioned media. ORY1001 treatment rescued expression of reactive astrogliosis-linked genes in HMG20A ablated astrocytes while enhancing cell surface area, GFAP intensity and STAT3 expression in healthy astrocytes, mimicking the effect of HMG20A. Furthermore, ORY1001 treatment protected against obesity-associated glucose intolerance in mice correlating with a regression of hypothalamic HMG20A expression, indicative of reactive astrogliosis attenuation with improved health status. Conclusion: HMG20A coordinates the astrocyte polarization state. Under physiological pressure such as obesity and insulin resistance that induces low grade inflammation, HMG20A expression is increased to induce reactive astrogliosis in an attempt to preserve the neuronal network and re-establish glucose homeostasis. Nonetheless, a chronic metabesity state or functional mutations will result in lower levels of HMG20A, failure to promote reactive astrogliosis and increase susceptibility of neurons to stress-induced apoptosis. Such effects could be reversed by ORY1001 treatment both in vitro and in vivo, paving the way for a new therapeutic approach for Type 2 Diabetes Mellitus.

Inhibition of the FACT Complex Targets Aberrant Hedgehog Signaling and Overcomes Resistance to Smoothened Antagonists.

Hedgehog signaling is aberrantly activated in hematologic malignancies and solid tumors, and targeting it is a promising therapeutic strategy against these cancers. Resistance to clinically available hedgehog-targeted Smoothened inhibitor (SMOi) drugs has become a critical issue in hedgehog-driven cancer treatment. Our previous studies identified inhibition of BET and CDK7 as two epigenetic/transcriptional-targeted therapeutic strategies for overcoming SMOi resistance, providing a promising direction for anti-hedgehog drug development. To uncover additional strategies for inhibiting aberrant hedgehog activity, here we performed CRISPR-Cas9 screening with an single-guide RNA library targeting epigenetic and transcriptional modulators in hedgehog-driven medulloblastoma cells, combined with tumor dataset analyses. Structure specific recognition protein 1 (SSRP1), a subunit of facilitates chromatin transcription (FACT) complex, was identified as a hedgehog-induced essential oncogene and therapeutic target in hedgehog-driven cancer. The FACT inhibitor CBL0137, which has entered clinical trials for cancer, effectively suppressed in vitro and in vivo growth of multiple SMOi-responsive and SMOi-resistant hedgehog-driven cancer models. Mechanistically, CBL0137 exerted anti-hedgehog activity by targeting transcription of GLI1 and GLI2, which are core transcription factors of the hedgehog pathway. SSRP1 bound the promoter regions of GLI1 and GLI2, while CBL0137 treatment substantially disrupted these interactions. Moreover, CBL0137 synergized with BET or CDK7 inhibitors to antagonize aberrant hedgehog pathway and growth of hedgehog-driven cancer models. Taken together, these results identify FACT inhibition as a promising epigenetic/transcriptional-targeted therapeutic strategy for treating hedgehog-driven cancers and overcoming SMOi resistance. SIGNIFICANCE: This study identifies FACT inhibition as an anti-hedgehog therapeutic strategy for overcoming resistance to Smoothened inhibitors and provides preclinical support for initiating clinical trials of FACT-targeted drug CBL0137 against hedgehog-driven cancers.

Effective inhibition of MYC-amplified group 3 medulloblastoma by FACT-targeted curaxin drug CBL0137.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor that can be categorized into four major molecular subgroups. Group 3 MB with MYC amplification (MYCamp-G3-MB) has been shown to be highly aggressive and exhibited worst prognosis, indicating the need for novel effective therapy most urgently. A few epigenetic targeted therapeutic strategies have recently been proven to effectively treat preclinical models of MYCamp-G3-MB, including BET inhibition, HDAC inhibition and SETD8 inhibition, unveiling a promising direction for further investigation. In this study, we carried out systemic bioinformatic analyses of public-available MB datasets as well as functional genomic screening datasets of primary MYCamp-G3-MB lines to search for other potential therapeutic targets within epigenetic modulators. We identified SSRP1, a subunit of histone-chaperone FACT complex, to be the top drug target candidate as it is highly cancer-dependent in whole-genome CRISPR-Cas9 screening across multiple MYCamp-G3-MB lines; significantly upregulated in MYCamp-G3-MB compared to normal cerebellum and most of the rest MB subtypes; its higher expression is correlated with worse prognosis; and it has a blood-brain-barrier penetrable targeted drug that has entered early phase human clinical trials already. Then we utilized RNA-interference approach to verify the cancer-dependency of SSRP1 in multiple MYCamp-G3-MB lines and further confirmed the therapeutic efficacy of FACT-targeted curaxin drug CBL0137 on treating preclinical models of MYCamp-G3-MB in vitro and in vivo, including an orthotopic intracranial xenograft model. Mechanistically, transcriptome analyses showed CBL0137 preferentially suppressed cell-cycle and DNA-repair related biological processes. Moreover, it selectively disrupted transcription of MYC and NEUROD1, two critical oncogenic transcription factors of MYCamp-G3-MB, via depleting FACT complex from their promoter regions. In summary, our study demonstrates FACT-targeted CBL0137 works effectively on treating MYCamp-G3-MB, presenting another promising epigenetic-targeted therapeutic strategy against the most devastating form of MB.

Histone chaperone FACT complex mediates oxidative stress response to promote liver cancer progression.

OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.

Targeting Histone Chaperone FACT Complex Overcomes 5-Fluorouracil Resistance in Colon Cancer.

Fluorouracil (5-FU) remains a first-line chemotherapeutic agent for colorectal cancer. However, a subset of colorectal cancer patients who have defective mismatch-repair (dMMR) pathway show resistance to 5-FU. Here, we demonstrate that the efficacy of 5-FU in dMMR colorectal cancer cells is largely dependent on the DNA base excision repair (BER) pathway. Downregulation of APE1, a key enzyme in the BER pathway, decreases IC50 of 5-FU in dMMR colorectal cancer cells by 10-fold. Furthermore, we discover that the facilitates chromatin transcription (FACT) complex facilitates 5-FU repair in DNA via promoting the recruitment and acetylation of APE1 (AcAPE1) to damage sites in chromatin. Downregulation of FACT affects 5-FU damage repair in DNA and sensitizes dMMR colorectal cancer cells to 5-FU. Targeting the FACT complex with curaxins, a class of small molecules, significantly improves the 5-FU efficacy in dMMR colorectal cancer in vitro (∼50-fold decrease in IC50) and in vivo xenograft models. We show that primary tumor tissues of colorectal cancer patients have higher FACT and AcAPE1 levels compared with adjacent nontumor tissues. Additionally, there is a strong clinical correlation of FACT and AcAPE1 levels with colorectal cancer patients' response to chemotherapy. Together, our study demonstrates that targeting FACT with curaxins is a promising strategy to overcome 5-FU resistance in dMMR colorectal cancer patients.

Computer-Aided Discovery of Small Molecule Inhibitors of Thymocyte Selection-Associated High Mobility Group Box Protein (TOX) as Potential Therapeutics for Cutaneous T-Cell Lymphomas.

Cutaneous T-cell lymphomas (CTCL) are the most common primary lymphomas of the skin. We have previously identified thymocyte selection-associated high mobility group (HMG) box protein (TOX) as a promising drug target in CTCL; however, there are currently no small molecules able to directly inhibit TOX. We aimed to address this unmet opportunity by developing anti-TOX therapeutics with the use of computer-aided drug discovery methods. The available NMR-resolved structure of the TOX protein was used to model its DNA-binding HMG-box domain. To investigate the druggability of the corresponding protein-DNA interface on TOX, we performed a pilot virtual screening of 200,000 small molecules using in silico docking and identified 'hot spots' for drug-binding on the HMG-box domain. We then performed a large-scale virtual screening of 7.6 million drug-like compounds that were available from the ZINC15 database. As a result, a total of 140 top candidate compounds were selected for subsequent in vitro validation. Of those, 18 small molecules have been characterized as selective TOX inhibitors.

The histone chaperone complex FACT promotes proliferative switch of G0 cancer cells.

Cancer cell repopulation through cell cycle re-entry by quiescent (G0 ) cell is thought to be an important mechanism behind treatment failure and cancer recurrence. Facilitates Chromatin Transcription (FACT) is involved in DNA repair, replication and transcription by eviction of histones or loosening their contact with DNA. While FACT expression is known to be high in a range of cancers, the biological significance of the aberrant increase is not clear. We found that in prostate and lung cancer cells FACT mRNA and protein levels were low at G0 compared to the proliferating state but replenished upon cell cycle re-entry. Silencing of FACT with Dox-inducible shRNA hindered cell cycle re-entry by G0 cancer cells, which could be rescued by ectopic expression of FACT. An increase in SKP2, c-MYC and PIRH2 and a decrease in p27 protein levels seen upon cell cycle re-entry were prevented or diminished when FACT was silenced. Further, using mVenus-p27K- infected cancer cells to measure p27 degradation capacity, we confirm that inhibition of FACT at release from quiescence suppressed the p27 degradation capacity resulting in an increased mVenus-p27K- signal. In conclusion, FACT plays an important role in promoting the transition from G0 to the proliferative state and can be a potential therapeutic target to prevent prostate and lung cancer from progression and recurrence.

The FACT inhibitor CBL0137 Synergizes with Cisplatin in Small-Cell Lung Cancer by Increasing NOTCH1 Expression and Targeting Tumor-Initiating Cells.

Traditional treatments of small-cell lung cancer (SCLC) with cisplatin, a standard-of-care therapy, spare the tumor-initiating cells (TIC) that mediate drug resistance. Here we report a novel therapeutic strategy that preferentially targets TICs in SCLC, in which cisplatin is combined with CBL0137, an inhibitor of the histone chaperone facilitates chromatin transcription (FACT), which is highly expressed in TICs. Combination of cisplatin and CBL0137 killed patient-derived and murine SCLC cell lines synergistically. In response to CBL0137 alone, TICs were more sensitive than non-TICs, in part, because CBL0137 increased expression of the tumor suppressor NOTCH1 by abrogating the binding of negative regulator SP3 to the NOTCH1 promoter, and in part because treatment decreased the high expression of stem cell transcription factors. The combination of cisplatin and CBL0137 greatly reduced the growth of a patient-derived xenograft in mice and also the growth of a syngeneic mouse SCLC tumor. Thus, CBL0137 can be a highly effective drug against SCLC, especially in combination with cisplatin.Significance: These findings reveal a novel therapeutic regimen for SCLC, combining cisplatin with an inhibitor that preferentially targets tumor-initiating cells. Cancer Res; 78(9); 2396-406. ©2018 AACR.

Anticancer drug candidate CBL0137, which inhibits histone chaperone FACT, is efficacious in preclinical orthotopic models of temozolomide-responsive and -resistant glioblastoma.

Background: The survival rate for patients with glioblastoma (GBM) remains dismal. New therapies targeting molecular pathways dysregulated in GBM are needed. One such clinical-stage drug candidate, CBL0137, is a curaxin, small molecules which simultaneously downregulate nuclear factor-kappaB (NF-ĸB) and activate p53 by inactivating the chromatin remodeling complex, Facilitates Chromatin Transcription (FACT). Methods: We used publicly available databases to establish levels of FACT subunit expression in GBM. In vitro, we evaluated the toxicity and effect of CBL0137 on FACT, p53, and NF-ĸB on U87MG and A1207 human GBM cells. In vivo, we implanted the cells orthotopically in nude mice and administered CBL0137 in various dosing regimens to assess brain and tumor accumulation of CBL0137, its effect on tumor cell proliferation and apoptosis, and on survival of mice with and without temozolomide (TMZ). Results: FACT subunit expression was elevated in GBM compared with normal brain. CBL0137 induced loss of chromatin-unbound FACT, activated p53, inhibited NF-ĸB-dependent transcription, and was toxic to GBM cells. The drug penetrated the blood-brain barrier and accumulated in orthotopic tumors significantly more than normal brain tissue. It increased apoptosis and suppressed proliferation in both U87MG and A1207 tumors. Intravenous administration of CBL0137 significantly increased survival in models of early- through late-stage TMZ-responsive and -resistant GBM, with a trend toward significantly increasing the effect of TMZ in TMZ-responsive U87MG tumors. Conclusion: CBL0137 targets GBM according to its proposed mechanism of action, crosses the blood-brain barrier, and is efficacious in both TMZ-responsive and -resistant orthotopic models, making it an attractive new therapy for GBM.

Multimodal MRI Evaluation of the MitoPark Mouse Model of Parkinson's Disease.

The MitoPark mouse, a relatively new genetic model of Parkinson's disease (PD), has a dopaminergic neuron-specific knock-out that inactivates the mitochondrial transcription factor A (Tfam), a protein essential for mitochondrial DNA expression and maintenance. This study used multimodal MRI to characterize the neuroanatomical correlates of PD-related deficits in MitoPark mice, along with functional behavioral tests. Compared with age-matched wild-type animals, MitoPark mice at 30 weeks showed: i) reduced whole-brain volume and increased ventricular volume, indicative of brain atrophy, ii) reduced transverse relaxation time (T2*) of the substantia nigra and striatum, suggestive of abnormal iron accumulation, iii) reduced apparent diffusion coefficient in the substantia nigra, suggestive of neuronal loss, iv) reduced fractional anisotropy in the corpus callosum and substantia nigra, indicative of white-matter damages, v) cerebral blood flow was not significantly affected, and vi) reduced motor activity in open-field tests, reduced memory in novel object recognition tests, as well as decreased mobility in tail suspension tests, an indication of depression. In sum, MitoPark mice recapitulate changes in many MRI parameters reported in PD patients. Multimodal MRI may prove useful for evaluating neuroanatomical correlates of PD pathophysiology in MitoPark mice, and for longitudinally monitoring disease progression and therapeutic interventions for PD.

Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma.

Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. We used a MYC target gene signature that predicts poor neuroblastoma prognosis to identify the histone chaperone FACT (facilitates chromatin transcription) as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small-molecule curaxin compound CBL0137 markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with standard chemotherapy by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells and suggesting a treatment strategy for MYCN-driven neuroblastoma.

FACT Proteins, SUPT16H and SSRP1, Are Transcriptional Suppressors of HIV-1 and HTLV-1 That Facilitate Viral Latency.

Our functional genomic RNAi screens have identified the protein components of the FACT (facilitates chromatin transcription) complex, SUPT16H and SSRP1, as top host factors that negatively regulate HIV-1 replication. FACT interacts specifically with histones H2A/H2B to affect assembly and disassembly of nucleosomes, as well as transcription elongation. We further investigated the suppressive role of FACT proteins in HIV-1 transcription. First, depletion of SUPT16H or SSRP1 protein enhances Tat-mediated HIV-1 LTR (long terminal repeat) promoter activity. Second, HIV-1 Tat interacts with SUPT16H but not SSRP1 protein. However, both SUPT16H and SSRP1 are recruited to LTR promoter. Third, the presence of SUPT16H interferes with the association of Cyclin T1 (CCNT1), a subunit of P-TEFb, with the Tat-LTR axis. Removing inhibitory mechanisms to permit HIV-1 transcription is an initial and key regulatory step to reverse post-integrated latent HIV-1 proviruses for purging of reservoir cells. We therefore evaluated the role of FACT proteins in HIV-1 latency and reactivation. Depletion of SUPT16H or SSRP1 protein affects both HIV-1 transcriptional initiation and elongation and spontaneously reverses latent HIV-1 in U1/HIV and J-LAT cells. Similar effects were observed with a primary CD4+ T cell model of HIV-1 latency. FACT proteins also interfere with HTLV-1 Tax-LTR-mediated transcription and viral latency, indicating that they may act as general transcriptional suppressors for retroviruses. We conclude that FACT proteins SUPT16H and SSRP1 play a key role in suppressing HIV-1 transcription and promoting viral latency, which may serve as promising gene targets for developing novel HIV-1 latency-reversing agents.

Evidence of an oncogenic role of aberrant TOX activation in cutaneous T-cell lymphoma.

TOX is a nuclear factor essential for the development of CD4(+) T cells in the thymus. It is normally expressed in low amounts in mature CD4(+) T cells of the skin and the peripheral blood. We have recently discovered that the transcript levels of TOX were significantly increased in mycosis fungoides, the most common type of cutaneous T-cell lymphoma (CTCL), as compared to normal skin or benign inflammatory dermatoses. However, its involvement in advanced CTCL and its biological effects on CTCL pathogenesis have not been explored. In this study, we demonstrate that TOX expression is also enhanced significantly in primary CD4(+)CD7(-) cells from patients with Sézary syndrome, a leukemic variant of CTCL, and that high TOX transcript levels correlate with increased disease-specific mortality. Stable knockdown of TOX in CTCL cells promoted apoptosis and reduced cell cycle progression, leading to less cell viability and colony-forming ability in vitro and to reduced tumor growth in vivo. Furthermore, TOX knockdown significantly increased 2 cyclin-dependent kinase (CDK) inhibitors, CDKN1B and CDKN1C. Lastly, blocking CDKN1B and CDKN1C reversed growth inhibition of TOX knockdown. Collectively, these findings provide strong evidence that aberrant TOX activation is a critical oncogenic event for CTCL.

Age-related mitochondrial DNA depletion and the impact on pancreatic Beta cell function.

Type 2 diabetes is characterised by an age-related decline in insulin secretion. We previously identified a 50% age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. The purpose of this study was to mimic this degree of mtDNA depletion in MIN6 cells to determine whether there is a direct impact on insulin secretion. Transcriptional silencing of mitochondrial transcription factor A, TFAM, decreased mtDNA levels by 40% in MIN6 cells. This level of mtDNA depletion significantly decreased mtDNA gene transcription and translation, resulting in reduced mitochondrial respiratory capacity and ATP production. Glucose-stimulated insulin secretion was impaired following partial mtDNA depletion, but was normalised following treatment with glibenclamide. This confirms that the deficit in the insulin secretory pathway precedes K+ channel closure, indicating that the impact of mtDNA depletion is at the level of mitochondrial respiration. In conclusion, partial mtDNA depletion to a degree comparable to that seen in aged human islets impaired mitochondrial function and directly decreased insulin secretion. Using our model of partial mtDNA depletion following targeted gene silencing of TFAM, we have managed to mimic the degree of mtDNA depletion observed in aged human islets, and have shown how this correlates with impaired insulin secretion. We therefore predict that the age-related mtDNA depletion in human islets is not simply a biomarker of the aging process, but will contribute to the age-related risk of type 2 diabetes.

The expression of the tumour suppressor HBP1 is down-regulated by growth factors via the PI3K/PKB/FOXO pathway.

Growth factors inactivate the FOXO (forkhead box O) transcription factors through PI3K (phosphoinositide 3-kinase) and PKB (protein kinase B). By comparing microarray data from multiple model systems, we identified HBP1 (high-mobility group-box protein 1) as a novel downstream target of this pathway. HBP1 mRNA was down-regulated by PDGF (platelet-derived growth factor), FGF (fibroblast growth factor), PI3K and PKB, whereas it was up-regulated by FOXO factors. This observation was confirmed in human and murine fibroblasts as well as in cell lines derived from leukaemia, breast adenocarcinoma and colon carcinoma. Bioinformatics analysis led to the identification of a conserved consensus FOXO-binding site in the HBP1 promoter. By luciferase activity assay and ChIP, we demonstrated that FOXO bound to this site and regulated the HBP1 promoter activity in a PI3K-dependent manner. Silencing of HBP1 by shRNA increased the proliferation of human fibroblasts in response to growth factors, suggesting that HBP1 limits cell growth. Finally, by analysing a transcriptomics dataset from The Cancer Genome Atlas, we observed that HBP1 expression was lower in breast tumours that had lost FOXO expression. In conclusion, HBP1 is a novel target of the PI3K/FOXO pathway and controls cell proliferation in response to growth factors.