Docetaxel-Induced Cell Death Is Regulated by a Fatty Acid-Binding Protein 12-Slug-Survivin Pathway in Prostate Cancer Cells.

Chemotherapy is an important treatment option for advanced prostate cancer, especially for metastatic prostate cancer (PCa). Resistance to first-line chemotherapeutic drugs such as docetaxel often accompanies prostate cancer progression. Attempts to overcome resistance to docetaxel by combining docetaxel with other biological agents have been mostly unsuccessful. A better understanding of the mechanisms underlying docetaxel resistance may provide new avenues for the treatment of advanced PCa. We have previously found that the fatty acid-binding protein 12 (FABP12)-PPARγ pathway modulates lipid-related bioenergetics and PCa metastatic transformation through induction of Slug, a master driver of epithelial-to-mesenchymal transition (EMT). Here, we report that the FABP12-Slug axis also underlies chemoresistance in PCa cells. Cell sensitivity to docetaxel is markedly suppressed in FABP12-expressing cells, along with induction of Survivin, a typical apoptosis inhibitor, and inhibition of cleaved PARP, a hallmark of programmed cell death. Importantly, Slug depletion down-regulates Survivin and restores cell sensitivity to docetaxel in FABP12-expressing cells. Finally, we also show that high levels of Survivin are associated with poor prognosis in PCa patients, with FABP12 status determining its prognostic significance. Our research identifies a FABP12-Slug-Survivin pathway driving docetaxel resistance in PCa cells, suggesting that targeting FABP12 may be a precision approach to improve chemodrug efficacy and curb metastatic progression in PCa.

Overcoming retinoic acid resistance in HER2-enriched breast cancers: role of MYC.

HER2-enriched (HER2+) breast cancers express high levels of the growth-promoting HER2 protein. Although these cancers are treated with the HER2-targeted drug, trastuzumab, resistance to treatment is common. Retinoic acid (RA) is an anti-cancer agent that has been successfully used for the treatment of leukemia and holds promise for the treatment of solid cancers, including breast cancer. The HER2 gene is frequently co-amplified with RARA, a key determinant of RA sensitivity in breast cancers. It seems surprising, therefore, that HER2+ breast cancers are refractory to RA treatment. Here, we show that MYC mediates RA resistance by suppressing the expression of cellular retinoic acid binding protein 2 (CRABP2), resulting in RARα inactivation. CRABP2 is an intracellular RA transporter that delivers RA to the nuclear receptor RARα for its activation. Our results indicate that response to RA is enhanced by MYC depletion in HER2+ breast cancer cells and that RA treatment enhances trastuzumab responsiveness. Our findings support the use of RA and trastuzumab for the treatment of subsets of patients with breast cancers that are HER2-RARα co-amplified and have low levels of MYC.

Role of DDX1 in the oxidative response of ataxia telangiectasia patient-derived fibroblasts.

Ataxia Telangiectasia (A-T) is an inherited autosomal recessive disorder characterized by cerebellar neurodegeneration, radiosensitivity, immunodeficiency and a high incidence of lymphomas. A-T is caused by mutations in the ATM gene. While loss of ATM function in DNA repair explains some aspects of A-T pathophysiology such as radiosensitivity and cancer predisposition, other A-T features such as neurodegeneration imply additional roles for ATM outside the nucleus. Emerging evidence suggests that ATM participates in cellular response to oxidative stress, failure of which contributes to the neurodegeneration associated with A-T. Here, we use fibroblasts derived from A-T patients to investigate whether DEAD Box 1 (DDX1), an RNA binding/unwinding protein that functions downstream of ATM in DNA double strand break repair, also plays a role in ATM-dependent cellular response to oxidative stress. Focusing on DDX1 target RNAs that are associated with neurological disorders and oxidative stress response, we show that ATM is required for increased binding of DDX1 to its target RNAs in the presence of arsenite-induced oxidative stress. Our results indicate that DDX1 functions downstream of ATM by protecting specific mRNAs in the cytoplasm of arsenite-treated cells. In keeping with a role for ATM and DDX1 in oxidative stress, levels of reactive oxygen species (ROS) are increased in ATM-deficient as well as DDX1-depleted cells. We propose that reduced levels of cytoplasmic DDX1 RNA targets sensitizes ATM-deficient cells to oxidative stress resulting in increased cell death. This sensitization would be especially detrimental to long-lived highly metabolically active cells such as neurons providing a possible explanation for the neurodegenerative defects associated with A-T.

Stationary-to-migratory transition in glioblastoma stem-like cells driven by a fatty acid-binding protein 7-RXRα neurogenic pathway.

BACKGROUND: Glioblastoma (GBM) stem-like cells (GSCs) are crucial drivers of treatment resistance and tumor recurrence. While the concept of "migrating" cancer stem cells was proposed a decade ago, the roles and underlying mechanisms of the heterogeneous populations of GSCs remain poorly defined. METHODS: Cell migration using GBM cell lines and patient-derived GSCs was examined using Transwell inserts and the scratch assay. Single-cell RNA sequencing data analysis were used to map GSC drivers to specific GBM cell populations. Xenografted mice were used to model the role of brain-type fatty acid-binding protein 7 (FABP7) in GBM infiltration and expansion. The mechanism by which FABP7 and its fatty acid ligands promote GSC migration was examined by gel shift and luciferase gene reporter assays. RESULTS: A subpopulation of FABP7-expressing migratory GSCs was identified, with FABP7 upregulating SOX2, a key modulator for GBM stemness and plasticity, and ZEB1, a prominent factor in GBM epithelial-mesenchymal transition and invasiveness. Our data indicate that GSC migration is driven by nuclear FABP7 through activation of RXRα, a nuclear receptor activated by polyunsaturated fatty acids (PUFAs). CONCLUSION: Infiltrative progression in GBM is driven by migratory GSCs through activation of a PUFA-FABP7-RXRα neurogenic pathway.

Radiation-Induced Cellular Senescence Reduces Susceptibility of Glioblastoma Cells to Oncolytic Vaccinia Virus.

Glioblastoma (GBM) is a malignant brain cancer refractory to the current standard of care, prompting an extensive search for novel strategies to improve outcomes. One approach under investigation is oncolytic virus (OV) therapy in combination with radiotherapy. In addition to the direct cytocidal effects of radiotherapy, radiation induces cellular senescence in GBM cells. Senescent cells cease proliferation but remain viable and are implicated in promoting tumor progression. The interaction of viruses with senescent cells is nuanced; some viruses exploit the senescent state to their benefit, while others are hampered, indicating senescence-associated antiviral activity. It is unknown how radiation-induced cellular senescence may impact the oncolytic properties of OVs based on the vaccinia virus (VACV) that are used in combination with radiotherapy. To better understand this, we induced cellular senescence by treating GBM cells with radiation, and then evaluated the growth kinetics, infectivity, and cytotoxicity of an oncolytic VACV, ∆F4LΔJ2R, as well as wild-type VACV in irradiated senescence-enriched and non-irradiated human GBM cell lines. Our results show that both viruses display attenuated oncolytic activities in irradiated senescence-enriched GBM cell populations compared to non-irradiated controls. These findings indicate that radiation-induced cellular senescence is associated with antiviral activity and highlight important considerations for the combination of VACV-based oncolytic therapies with senescence-inducing agents such as radiotherapy.

RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. The standard treatment achieves a median overall survival for GBM patients of only 15 months. Hence, novel therapies based on an increased understanding of the mechanistic underpinnings of GBM are desperately needed. In this study, we show that elevated expression of 28S rRNA (cytosine-C(5))-methyltransferase NSUN5, which methylates cytosine 3782 of 28S rRNA in GBM cells, is strongly associated with the poor survival of GBM patients. Moreover, we demonstrate that overexpression of NSUN5 increases protein synthesis in GBM cells. NSUN5 knockdown decreased protein synthesis, cell proliferation, sphere formation, migration, and resistance to temozolomide in GBM cell lines. NSUN5 knockdown also decreased the number and size of GBM neurospheres in vitro. As a corollary, mice harboring U251 tumors wherein NSUN5 was knocked down survived longer than mice harboring control tumors. Taken together, our results suggest that NSUN5 plays a protumorigenic role in GBM by enabling the enhanced protein synthesis requisite for tumor progression. Accordingly, NSUN5 may be a hitherto unappreciated target for the treatment of GBM.

Radiation combined with oncolytic vaccinia virus provides pronounced antitumor efficacy and induces immune protection in an aggressive glioblastoma model.

Glioblastoma (GB) is a malignant and immune-suppressed brain cancer that remains incurable despite the current standard of care. Radiotherapy is a mainstay of GB treatment, however invasive cancer cells outside the irradiated field and radioresistance preclude complete eradication of GB cells. Oncolytic virus therapy harnesses tumor-selective viruses to spread through and destroy tumors while stimulating antitumor immune responses, and thus has potential for use following radiotherapy. We demonstrate that oncolytic ΔF4LΔJ2R vaccinia virus (VACV) replicates in and induces cytotoxicity of irradiated brain tumor initiating cells in vitro. Importantly, a single 10 Gy dose of radiation combined with ΔF4LΔJ2R VACV produced considerably superior anticancer effects relative to either monotherapy when treating immune-competent orthotopic CT2A-luc mouse models-significantly extending survival and curing the majority of mice. Mice cured by the combination displayed significantly increased survival relative to naïve age-matched controls following intracranial tumor challenge, with some complete rejections. Further, the combination therapy was associated with an increased ratio of CD8+ effector T cells to regulatory T cells compared to either monotherapy. This study validates the use of radiation with an oncolytic ΔF4LΔJ2R VACV to improve treatment of this malignant brain cancer.

Lipid phosphate phosphatase-2 promotes tumor growth through increased c-Myc expression.

LPP2 is one of three enzymes in the lipid phosphate phosphatase family (LPP1-3) that dephosphorylate extracellular and intracellular bioactive lipid phosphates and pyrophosphates. LPP2 increases cell growth and LPP2 expression is elevated in a variety of malignancies, implying that LPP2 is a pro-tumorigenic factor. Methods: LPP2 expression in human breast tumors and normal breast tissue was measured by qPCR. To understand the role of LPP2, we knocked out its expression in multiple cell lines using CRISPR/Cas9. Cell proliferation and migration were compared between wild type and LPP2 knockout cells. Cell cycle was measured by flow cytometry, and cell cycle proteins were determined by western blotting. Effects of LPP2 on tumor growth were investigated using syngeneic and xenograft mouse breast cancer models. Results: LPP2 mRNA levels were higher in ER/PR positive, ER/HER2 positive, and triple negative human breast tumors, relative to normal breast tissue. Higher levels of LPP2 in breast tumors, hepatocellular carcinoma, pancreatic adenocarcinoma, and melanomas were prognostic of poorer survival. LPP2 mRNA expression is also increased in Hs-578T, MDA-MB-231, MCF7 and MDA-MB-468 breast cancer cell lines, relative to non-malignant Hs-578Bst, MCF10A and MCF-12A cells. LPP2 knockout in breast cancer cells decreased cell growth by inhibiting G1/S transition, whereas, increasing LPP2 levels in Hs-578Bst and MCF10A cells promoted proliferation. The effects of LPP2 on cell cycle were associated with changes in cyclin A2, cyclin B1, and cell cycle inhibitors, p27 or p21. The level of c-Myc was downregulated by knocking out LPP2, and it was partly restored by re-expressing LPP2. The positive correlation between the expression of LPP2 and c-Myc exists in multiple cancer cell lines including breast, lung, upper aerodigestive tract and urinary tract cancer. LPP2 knockout in MDA-MB-231 or 4T1 cells suppressed tumor formation in mouse breast cancer models, and decreased the in vivo expression of Ki67 and c-Myc of the cancer cells. Conclusion: Targeting LPP2 could provide a new strategy for decreasing c-Myc expression and tumor growth.

DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos.

The DEAD box protein DDX1, previously associated with 3'-end RNA processing and DNA repair, forms large aggregates in the cytoplasm of early mouse embryos. Ddx1 knockout causes stalling of embryos at the 2-4 cell stages. Here, we identify a DDX1-containing membrane-bound calcium-containing organelle with a nucleic acid core. We show that aggregates of these organelles form ring-like structures in early-stage embryos which we have named Membrane Associated RNA-containing Vesicles. We present evidence that DDX1 is required for the formation of Membrane Associated RNA-containing Vesicles which in turn regulate the spatial distribution of calcium in embryos. We find that Ddx1 knockout in early embryos disrupts calcium distribution, and increases mitochondria membrane potential, mitochondrial activity, and reactive oxygen species. Sequencing analysis of embryos from Ddx1 heterozygote crosses reveals downregulation of a subset of RNAs involved in developmental and mitochondrial processes in the embryos with low Ddx1 RNA. We propose a role for Membrane Associated RNA-containing Vesicles in calcium-controlled mitochondrial functions that are essential for embryonic development.

DEAD box 1 (DDX1) protein binds to and protects cytoplasmic stress response mRNAs in cells exposed to oxidative stress.

The integrated stress response is a network of highly orchestrated pathways activated when cells are exposed to environmental stressors. While global repression of translation is a well-recognized hallmark of the integrated stress response, less is known about the regulation of mRNA stability during stress. DEAD box proteins are a family of RNA unwinding/remodeling enzymes involved in every aspect of RNA metabolism. We previously showed that DEAD box 1 (DDX1) protein accumulates at DNA double-strand breaks during genotoxic stress and promotes DNA double-strand break repair via homologous recombination. Here, we examine the role of DDX1 in response to environmental stress. We show that DDX1 is recruited to stress granules (SGs) in cells exposed to a variety of environmental stressors, including arsenite, hydrogen peroxide, and thapsigargin. We also show that DDX1 depletion delays resolution of arsenite-induced SGs. Using RNA immunoprecipitation sequencing, we identify RNA targets bound to endogenous DDX1, including RNAs transcribed from genes previously implicated in stress responses. We show the amount of target RNAs bound to DDX1 increases when cells are exposed to stress, and the overall levels of these RNAs are increased during stress in a DDX1-dependent manner. Even though DDX1's RNA-binding property is critical for maintenance of its target mRNA levels, we found RNA binding is not required for localization of DDX1 to SGs. Furthermore, DDX1 knockdown does not appear to affect RNA localization to SGs. Taken together, our results reveal a novel role for DDX1 in maintaining cytoplasmic mRNA levels in cells exposed to oxidative stress.

FABP7 Facilitates Uptake of Docosahexaenoic Acid in Glioblastoma Neural Stem-like Cells.

Glioblastoma (GBM) is an aggressive tumor with a dismal prognosis. Neural stem-like cells contribute to GBM's poor prognosis by driving drug resistance and maintaining cellular heterogeneity. GBM neural stem-like cells express high levels of brain fatty acid-binding protein (FABP7), which binds to polyunsaturated fatty acids (PUFAs) ω-6 arachidonic acid (AA) and ω-3 docosahexaenoic acid (DHA). Similar to brain, GBM tissue is enriched in AA and DHA. However, DHA levels are considerably lower in GBM tissue compared to adult brain. Therefore, it is possible that increasing DHA content in GBM, particularly in neural stem-like cells, might have therapeutic value. Here, we examine the fatty acid composition of patient-derived GBM neural stem-like cells grown as neurosphere cultures. We also investigate the effect of AA and DHA treatment on the fatty acid profiles of GBM neural stem-like cells with or without FABP7 knockdown. We show that DHA treatment increases DHA levels and the DHA:AA ratio in GBM neural stem-like cells, with FABP7 facilitating the DHA uptake. We also found that an increased uptake of DHA inhibits the migration of GBM neural stem-like cells. Our results suggest that increasing DHA content in the GBM microenvironment may reduce the migration/infiltration of FABP7-expressing neural stem-like cancer cells.

Super resolution microscopy reveals DHA-dependent alterations in glioblastoma membrane remodelling and cell migration.

Brain fatty acid binding protein (FABP7; B-FABP) promotes glioblastoma (GBM) cell migration and is associated with tumor infiltration, properties associated with a poor prognosis in GBM patients. FABP7-expressing neural stem-like cells are known to drive tumor migration/infiltration and resistance to treatment. We have previously shown that FABP7's effects on cell migration can be reversed when GBM cells are cultured in medium supplemented with the omega-3 fatty acid, docosahexaenoic acid (DHA). Here, we use super-resolution imaging on patient-derived GBM stem-like cells to examine the importance of FABP7 and its fatty acid ligands in mitigating GBM cell migration. As FABPs are involved in fatty acid transport from membrane to cytosol, we focus on the effect of FABP7 and its ligand DHA on GBM membrane remodeling, as well as FABP7 nanoscale domain formation on GBM membrane. Using quantitative plasma membrane lipid order imaging, we show that FABP7 expression in GBM cells correlates with increased membrane lipid order, with DHA dramatically decreasing lipid order. Using super-resolution stimulated emission depletion (STED) microscopy, we observe non-uniform distribution of FABP7 on the surface of GBM cells, with FABP7 forming punctate nanoscale domains of ∼100 nm in diameter. These nanodomains are particularly enriched at the migrating front of GBM cells. Interestingly, FABP7 nanodomains are disrupted when GBM cells are cultured in DHA-supplemented medium. We demonstrate a tight link between cell migration, a higher membrane lipid order and increased FABP7 nanoscale domains. We propose that DHA-mediated disruption of membrane lipid order and FABP7 nanodomains forms the basis of FABP7/DHA-mediated inhibition of cell migration in GBM.

An Amplified Fatty Acid-Binding Protein Gene Cluster in Prostate Cancer: Emerging Roles in Lipid Metabolism and Metastasis.

Treatment for early stage and localized prostate cancer (PCa) is highly effective. Patient survival, however, drops dramatically upon metastasis due to drug resistance and cancer recurrence. The molecular mechanisms underlying PCa metastasis are complex and remain unclear. It is therefore crucial to decipher the key genetic alterations and relevant molecular pathways driving PCa metastatic progression so that predictive biomarkers and precise therapeutic targets can be developed. Through PCa cohort analysis, we found that a fatty acid-binding protein (FABP) gene cluster (containing five FABP family members) is preferentially amplified and overexpressed in metastatic PCa. All five FABP genes reside on chromosome 8 at 8q21.13, a chromosomal region frequently amplified in PCa. There is emerging evidence that these FABPs promote metastasis through distinct biological actions and molecular pathways. In this review, we discuss how these FABPs may serve as drivers/promoters for PCa metastatic transformation using patient cohort analysis combined with a review of the literature.

The FABP12/PPARγ pathway promotes metastatic transformation by inducing epithelial-to-mesenchymal transition and lipid-derived energy production in prostate cancer cells.

Early stage localized prostate cancer (PCa) has an excellent prognosis; however, patient survival drops dramatically when PCa metastasizes. The molecular mechanisms underlying PCa metastasis are complex and remain unclear. Here, we examine the role of a new member of the fatty acid-binding protein (FABP) family, FABP12, in PCa progression. FABP12 is preferentially amplified and/or overexpressed in metastatic compared to primary tumors from both PCa patients and xenograft animal models. We show that FABP12 concurrently triggers metastatic phenotypes (induced epithelial-to-mesenchymal transition (EMT) leading to increased cell motility and invasion) and lipid bioenergetics (increased fatty acid uptake and accumulation, increased ATP production from fatty acid ß-oxidation) in PCa cells, supporting increased reliance on fatty acids for energy production. Mechanistically, we show that FABP12 is a driver of PPARγ activation which, in turn, regulates FABP12's role in lipid metabolism and PCa progression. Our results point to a novel role for a FABP-PPAR pathway in promoting PCa metastasis through induction of EMT and lipid bioenergetics.

Cytoplasmic aggregation of DDX1 in developing embryos: Early embryonic lethality associated with Ddx1 knockout.

Temporally-regulated maternal RNA translation is essential for embryonic development, with defective degradation resulting in stalled 2-cell embryos. We show that DDX1, a DEAD box protein implicated in RNA transport, may be a key regulator of maternal RNA utilization. DDX1 protein localizes exclusively to cytoplasmic granules in both oocytes and early stage mouse embryos, with DDX1 requiring RNA for retention at these sites. Homozygous knockout of Ddx1 causes stalling of mouse embryos at the 2-4 cell stages. These results suggest a maternal RNA-dependent role for DDX1 in the progression of embryos past the 2-4 cell stage. The change in appearance of DDX1-containing granules in developing embryos further supports a role in temporally-regulated degradation of RNAs. We carried out RNA-immunoprecipitations (RNA-IPs) to identify mRNAs bound to DDX1 in 2-cell embryos, focusing on 16 maternal genes previously shown to be essential for embryonic development past the 1- to 2-cell stages. Five of these RNAs were preferentially bound by DDX1: Ago2, Zar1, Tle6, Floped and Tif1α. We propose that DDX1 controls access to subsets of key maternal RNAs required for early embryonic development.

A positive feedback loop involving nuclear factor IB and calpain 1 suppresses glioblastoma cell migration.

Glioblastoma (GBM) is a brain tumor that remains largely incurable because of its highly-infiltrative properties. Nuclear factor I (NFI)-type transcription factors regulate genes associated with GBM cell migration and infiltration. We have previously shown that NFI activity depends on the NFI phosphorylation state and that calcineurin phosphatase dephosphorylates and activates NFI. Calcineurin is cleaved and activated by calpain proteases whose activity is, in turn, regulated by an endogenous inhibitor, calpastatin (CAST). The CAST gene is a target of NFI in GBM cells, with differentially phosphorylated NFIs regulating the levels of CAST transcript variants. Here, we uncovered an NFIB-calpain 1-positive feedback loop mediated through CAST and calcineurin. In NFI-hyperphosphorylated GBM cells, NFIB expression decreased the CAST-to-calpain 1 ratio in the cytoplasm. This reduced ratio increased autolysis and activity of cytoplasmic calpain 1. Conversely, in NFI-hypophosphorylated cells, NFIB expression induced differential subcellular compartmentalization of CAST and calpain 1, with CAST localizing primarily to the cytoplasm and calpain 1 to the nucleus. Overall, this altered compartmentalization increased nuclear calpain 1 activity. We also show that nuclear calpain 1, by cleaving and activating calcineurin, induces NFIB dephosphorylation. Of note, knockdown of calpain 1, NFIB, or both increased GBM cell migration and up-regulated the pro-migratory factors fatty acid-binding protein 7 (FABP7) and Ras homolog family member A (RHOA). In summary, our findings reveal bidirectional cross-talk between NFIB and calpain 1 in GBM cells. A physiological consequence of this positive feedback loop appears to be decreased GBM cell migration.

Non-canonical BAD activity regulates breast cancer cell and tumor growth via 14-3-3 binding and mitochondrial metabolism.

The Bcl-2-associated death promoter BAD is a prognostic indicator for good clinical outcome of breast cancer patients; however, whether BAD affects breast cancer biology is unknown. Here we showed that BAD increased cell growth in breast cancer cells through two distinct mechanisms. Phosphorylation of BAD at S118 increased S99 phosphorylation, 14-3-3 binding and AKT activation to promote growth and survival. Through a second, more prominent pathway, BAD stimulated mitochondrial oxygen consumption in a novel manner that was downstream of substrate entry into the mitochondria. BAD stimulated complex I activity that facilitated enhanced cell growth and sensitized cells to apoptosis in response to complex I blockade. We propose that this dependence on oxidative metabolism generated large but nonaggressive cancers. This model identifies a non-canonical role for BAD and reconciles BAD-mediated tumor growth with favorable outcomes in BAD-high breast cancer patients.

Effects of nuclear factor I phosphorylation on calpastatin (CAST) gene variant expression and subcellular distribution in malignant glioma cells.

Malignant glioma (MG) is the most lethal primary brain tumor. In addition to having inherent resistance to radiation treatment and chemotherapy, MG cells are highly infiltrative, rendering focal therapies ineffective. Genes involved in MG cell migration and glial cell differentiation are up-regulated by hypophosphorylated nuclear factor I (NFI), which is dephosphorylated by the phosphatase calcineurin in MG cells. Calcineurin is cleaved and thereby activated by calpain proteases, which are, in turn, inhibited by calpastatin (CAST). Here, we show that the CAST gene is a target of NFI and has NFI-binding sites in its intron 3 region. We also found that NFI-mediated regulation of CAST depends on NFI's phosphorylation state. We noted that occupation of CAST intron 3 by hypophosphorylated NFI results in increased activation of an alternative promoter. This activation resulted in higher levels of CAST transcript variants, leading to increased levels of CAST protein that lacks the N-terminal XL domain. CAST was primarily present in the cytoplasm of NFI-hypophosphorylated MG cells, with a predominantly perinuclear immunostaining pattern. NFI knockdown in NFI-hypophosphorylated MG cells increased CAST levels at the plasma membrane. These results suggest that NFI plays an integral role in the regulation of CAST variants and CAST subcellular distribution. Along with the previous findings indicating that NFI activity is regulated by calcineurin, these results provide a foundation for further investigations into the possibility of regulatory cross-talk between NFI and the CAST/calpain/calcineurin signaling pathway in MG cells.

NFIB promotes cell survival by directly suppressing p21 transcription in TP53-mutated triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited treatment options and poor prognosis. There is an urgent need to identify and understand the key factors and signalling pathways driving TNBC tumour progression, relapse, and treatment resistance. In this study, we report that gene copy numbers and expression levels of nuclear factor IB (NFIB), a recently identified oncogene in small cell lung cancer, are preferentially increased in TNBC compared to other breast cancer subtypes. Furthermore, increased levels of NFIB are significantly associated with high tumour grade, poor prognosis, and reduced chemotherapy response. Concurrent TP53 mutations and NFIB overexpression (z-scores > 0) were observed in 77.9% of TNBCs, in contrast to 28.5% in non-TNBCs. Depletion of NFIB in TP53-mutated TNBC cell lines promotes cell death, cell cycle arrest, and enhances sensitivity to docetaxel, a first-line chemotherapeutic drug in breast cancer treatment. Importantly, these alterations in growth properties were accompanied by induction of CDKN1A, the gene encoding p21, a downstream effector of p53. We show that NFIB directly interacts with the CDKN1A promoter in TNBC cells. Furthermore, knockdown of combined p21 and NFIB reverses the docetaxel-induced cell growth inhibition observed upon NFIB knockdown, indicating that NFIB's effect on chemotherapeutic drug response is mediated through p21. Our results indicate that NFIB is an important TNBC factor that drives tumour cell growth and drug resistance, leading to poor clinical outcomes. Thus, targeting NFIB in TP53-mutated TNBC may reverse oncogenic properties associated with mutant p53 by restoring p21 activity. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.