XAF1 destabilizes estrogen receptor α through the assembly of a BRCA1-mediated destruction complex and promotes estrogen-induced apoptosis.

X-linked inhibitor of apoptosis-associated factor 1 (XAF1) is a pro-apoptotic tumor suppressor that is frequently inactivated in multiple human cancers. However, its candidacy as a suppressor in the pathogenesis of breast cancer remains undefined. Here, we report that XAF1 acts as a molecular switch in estrogen (E2)-mediated cell-fate decisions favoring apoptosis over cell proliferation. XAF1 promoter hypermethylation is observed predominantly in estrogen receptor α (ERα)-positive versus ERα-negative tumor cells and associated with attenuated apoptotic response to E2. XAF1 is activated by E2 through a G protein-coupled estrogen receptor-mediated non-genomic pathway and induces ERα degradation and apoptosis while it is repressed by ERα for E2 stimulation of cell proliferation. The XAF1-ERα mutual antagonism dictates the outcomes of E2 signaling and its alteration is linked to the development of E2-resistant tumors. Mechanistically, XAF1 destabilizes ERα through the assembly of breast cancer-associated gene 1 (BRCA1)-mediated destruction complex. XAF1 interacts with ERα and BRCA1 via the zinc finger (ZF) domains 5/6 and 4, respectively, and the mutants lacking either of these domains fail to drive ERα ubiquitination and apoptosis. E2-induced regression of XAF1+/+ tumors is abolished by XAF1 depletion while XAF1-/- tumors recover E2 response by XAF1 restoration. XAF1 and ERα expression show an inverse correlation in primary breast tumors, and XAF1 expression is associated with the overall survival of patients with ERα-positive but not ERα-negative cancer. Together, this study uncovers an important role for the XAF1-ERα antagonism as a linchpin to govern E2-mediated cell-fate decisions, illuminating the mechanistic consequence of XAF1 alteration in breast tumorigenesis.

XAF1 drives apoptotic switch of endoplasmic reticulum stress response through destabilization of GRP78 and CHIP.

X-linked inhibitor of apoptosis-associated factor-1 (XAF1) is a stress-inducible tumor suppressor that is commonly inactivated in many human cancers. Despite accumulating evidence for the pro-apoptotic role for XAF1 under various stressful conditions, its involvement in endoplasmic reticulum (ER) stress response remains undefined. Here, we report that XAF1 increases cell sensitivity to ER stress and acts as a molecular switch in unfolded protein response (UPR)-mediated cell-fate decisions favoring apoptosis over adaptive autophagy. Mechanistically, XAF1 interacts with and destabilizes ER stress sensor GRP78 through the assembly of zinc finger protein 313 (ZNF313)-mediated destruction complex. Moreover, XAF1 expression is activated through PERK-Nrf2 signaling and destabilizes C-terminus of Hsc70-interacting protein (CHIP) ubiquitin E3 ligase, thereby blocking CHIP-mediated K63-linked ubiquitination and subsequent phosphorylation of inositol-required enzyme-1α (IRE1α) that is involved in in the adaptive ER stress response. In tumor xenograft assays, XAF1-/- tumors display substantially lower regression compared to XAF1+/+ tumors in response to cytotoxic dose of ER stress inducer. XAF1 and GRP78 expression show an inverse correlation in human cancer cell lines and primary breast carcinomas. Collectively this study uncovers an important role for XAF1 as a linchpin to govern the sensitivity to ER stress and the outcomes of UPR signaling, illuminating the mechanistic consequence of XAF1 inactivation in tumorigenesis.

XAF1 directs glioma response to temozolomide through apoptotic transition of autophagy by activation of ATM-AMPK signaling.

Background: X-linked inhibitor of apoptosis-associated factor 1 (XAF1) is a tumor suppressor that is commonly inactivated in multiple human cancers. However, its role in the pathogenesis and therapeutic response of glioma is poorly characterized. Methods: XAF1 activation by temozolomide (TMZ) and its effect on TMZ cytotoxicity were defined using luciferase reporter, flow cytometry, and immunofluorescence assays. Signaling mechanism was analyzed using genetic and pharmacologic experiments. In vivo studies were performed in mice to validate the role of XAF1 in TMZ therapy. Results: Epigenetic alteration of XAF1 is frequent in cell lines and primary tumors and contributes to cancer cell growth. XAF1 transcription is activated by TMZ via JNK-IRF-1 signaling to promote apoptosis while it is impaired by promoter hypermethylation. In tumor cells expressing high O 6-methylguanine-DNA methyltransferase (MGMT), XAF1 response to TMZ is debilitated. XAF1 facilitates TMZ-mediated autophagic flux to direct an apoptotic transition of protective autophagy. Mechanistically, XAF1 is translocated into the mitochondria to stimulate reactive oxygen species (ROS) production and ataxia telangiectasia mutated (ATM)-AMP-activated protein kinase (AMPK) signaling. A mutant XAF1 lacking the zinc finger 6 domain fails to localize in the mitochondria and activate ROS-ATM-AMPK signaling and autophagy-mediated apoptosis. XAF1-restored xenograft tumors display a reduced growth rate and enhanced therapeutic response to TMZ, which is accompanied with activation of ATM-AMPK signaling. XAF1 expression is associated with overall survival of TMZ treatment patients, particularly with low MGMT cancer. Conclusions: This study uncovers an important role for the XAF1-ATM-AMPK axis as a linchpin to govern glioma response to TMZ therapy.

NORE1A directs apoptotic switch of TNF signaling through reciprocal modulation of ITCH-mediated destruction of TNFRI and BAX.

NORE1A (RASSF5) is a tumor suppressor of the Ras-association domain family (RASSF) that is commonly inactivated in multiple human cancers. However, the molecular mechanism underlying its growth inhibition function remains largely undefined. Here we report that NORE1A antagonizes tumor necrosis factor receptor I (TNFRI) through the assembly of ITCH-mediated destruction complex to suppress TNF-NF-κB signaling and tumorigenesis. Moreover, NORE1A is identified as a transcription target of NF-κB, which directs an apoptotic switch of TNF effect by blocking ITCH interaction with and ubiquitination of BAX. Mechanistically, NORE1A binds directly to TNFRI and ITCH via the C1 and PPXY domains, respectively to facilitate the formation of ITCH-mediated destruction complex followed by ubiquitination-mediated lysosomal degradation of TNFRI. Through this function, NORE1A suppresses TNF-induced NF-κB-mediated transcription of pro-inflammatory and tumor-promoting genes, epithelial-to-mesenchymal transition, invasion and migration of tumor cells, and also debilitates tumor cell activation of macrophage and fibroblast. While NORE1A suppresses TNF receptor-mediated apoptosis, it activates TNF-induced apoptosis through BAX activation by protecting BAX from ITCH binding and ubiquitination. Cytotoxic response to TNF is substantially attenuated in NORE1A-depleted cells and tumors, and NORE1A-induced tumor regression is highly impeded in BAX-depleted tumors. An inverse correlation is shown between NORE1A and TNFRI expression in both cancer cell lines and primary tumors, and NORE1A effect on survival of cancer patients is strongly associated with expression status of ITCH. Collectively, this study uncovers that NORE1A directs a substrate switch of ITCH favoring TNFRI over BAX to terminate TNF signaling and accelerate apoptosis, illuminating the mechanistic consequence of NORE1A inactivation in tumorigenesis.

Mitochondrial relocation of a common synthetic antibiotic: A non-genotoxic approach to cancer therapy.

Tumor recurrence as a result of therapy-induced nuclear DNA lesions is a major issue in cancer treatment. Currently, only a few examples of potentially non-genotoxic drugs have been reported. Mitochondrial re-localization of ciprofloxacin, one of the most commonly prescribed synthetic antibiotics, is reported here as a new approach. Conjugating ciprofloxacin to a triphenyl phosphonium group (giving lead Mt-CFX), is used to enhance the concentration of ciprofloxacin in the mitochondria of cancer cells. The localization of Mt-CFX to the mitochondria induces oxidative damage to proteins, mtDNA, and lipids. A large bias in favor of mtDNA damage over nDNA was seen with Mt-CFX, contrary to classic cancer chemotherapeutics. Mt-CFX was found to reduce cancer growth in a xenograft mouse model and proved to be well tolerated. Mitochondrial relocalization of antibiotics could emerge as a useful approach to generating anticancer leads that promote cell death via the selective induction of mitochondrially-mediated oxidative damage.

XAF1 forms a positive feedback loop with IRF-1 to drive apoptotic stress response and suppress tumorigenesis.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a proapoptotic tumor suppressor that is frequently inactivated in multiple human cancers. However, the molecular basis for the XAF1-mediated growth inhibition remains largely undefined. Here, we report that XAF1 forms a positive feedback loop with interferon regulatory factor-1 (IRF-1) and functions as a transcriptional coactivator of IRF-1 to suppress tumorigenesis. Under various stressful conditions, XAF1 transcription is activated by IRF-1, and elevated XAF1 stabilizes and activates IRF-1. Mechanistically, XAF1 binds to the multifunctional domain 2 of IRF-1 via the zinc finger domain 6, thereby hindering C-terminus of Hsc70-interacting protein (CHIP) interaction with and ubiquitination of IRF-1. Activation of the IRF-1-XAF1 loop greatly increases stress-induced apoptosis and decreases the invasive capability of tumor cells. Oncogenic Ras and growth factors interfere with the IRF-1-XAF1 interplay via Erk-mediated repression of XAF1 transcription. Furthermore, XAF1 enhances IRF-1-mediated transcription of proapoptotic genes via the XAF1-IRF-1 complex formation on these target promoters. Meanwhile, XAF1 inhibits NF-κB-mediated tumor cell malignancy by reinforcing IRF-1 binding to a subset of coregulated promoters. Expression levels of IRF-1 and XAF1 correlate tightly in both cancer cell lines and primary tumors, and XAF1-induced tumor regression is markedly attenuated in IRF-1-depleted tumors. Collectively, this study identifies a novel mechanism of XAF1-mediated tumor suppression, uncovering XAF1 as a feedback coactivator of IRF-1 under stressful conditions.

PLK1-Targeted Fluorescent Tumor Imaging with High Signal-to-Background Ratio.

As significantly expressed during cell division, polo-like kinase 1 (PLK1) plays crucial roles in numerous mitotic events and has attracted interest as a potential therapeutic marker in oncological drug discovery. We prepared two small molecular fluorescent probes, 1 and 2, conjugated to SBE13 (a type II PLK1 inhibitor) to investigate the PLK1-targeted imaging of cancer cells and tumors. Enzymatic docking studies, molecular dynamics simulations, and in vitro and in vivo imaging experiments all supported the selective targeting and visualization of PLK1 expressing cells by probes 1 and 2, and probe 2 was successfully demonstrated to image PLK1-upregulated tumors with remarkable signal-to-background ratios. These findings represent the first example of small-molecule based fluorescent imaging of tumors using PLK1 as a target, which could provide new avenues for tumor diagnosis and precision therapeutics.

RASSF1A Directly Antagonizes RhoA Activity through the Assembly of a Smurf1-Mediated Destruction Complex to Suppress Tumorigenesis.

RASSF1A is a tumor suppressor implicated in many tumorigenic processes; however, the basis for its tumor suppressor functions are not fully understood. Here we show that RASSF1A is a novel antagonist of protumorigenic RhoA activity. Direct interaction between the C-terminal amino acids (256-277) of RASSF1A and active GTP-RhoA was critical for this antagonism. In addition, interaction between the N-terminal amino acids (69-82) of RASSF1A and the ubiquitin E3 ligase Smad ubiquitination regulatory factor 1 (Smurf1) disrupted GTPase activity by facilitating Smurf1-mediated ubiquitination of GTP-RhoA. We noted that the RhoA-binding domain of RASSF1A displayed high sequence homology with Rho-binding motifs in other RhoA effectors, such as Rhotekin. As predicted on this basis, RASSF1A competed with Rhotekin to bind RhoA and to block its activation. RASSF1A mutants unable to bind RhoA or Smurf1 failed to suppress RhoA-induced tumor cell proliferation, drug resistance, epithelial-mesenchymal transition, migration, invasion, and metastasis. Clinically, expression levels of RASSF1A and RhoA were inversely correlated in many types of primary and metastatic tumors and tumor cell lines. Collectively, our findings showed how RASSF1A may suppress tumorigenesis by intrinsically inhibiting the tumor-promoting activity of RhoA, thereby illuminating the potential mechanistic consequences of RASSF1A inactivation in many cancers. Cancer Res; 76(7); 1847-59. ©2016 AACR.

PPARδ promotes oncogenic redirection of TGF-ß1 signaling through the activation of the ABCA1-Cav1 pathway.

TGF-ß1 plays biphasic functions in prostate tumorigenesis, inhibiting cell growth at early stages but promoting malignant progression at later stages. However, the molecular basis for the oncogenic conversion of TGF-ß1 function remains largely undefined. Here, we demonstrate that PPARδ is a direct transcription target of TGF-ß1 and plays a critical role in oncogenic redirection of TGF-ß1 signaling. Blockade of PPARδ induction enhances tumor cell response to TGF-ß1-mediated growth inhibition, while its activation promotes TGF-ß1-induced tumor growth, migration and invasion. PPARδ-mediated switch of TGF-ß1 function is associated with down- and upregulation of Smad and ERK signaling, respectively, and tightly linked to its function to activate ABCA1 cholesterol transporter followed by caveolin-1 (Cav1) induction. Intriguingly, TGF-ß1 activation of the PPARδ-ABCA1-Cav1 pathway facilitates degradation of TGF-ß receptors (TßRs) and attenuates Smad but enhances ERK response to TGF-ß1. Expression of PPARδ and Cav1 is tightly correlated in both prostate tissues and cell lines and significantly higher in cancer vs. normal tissues. Collectively, our study shows that PPARδ is a transcription target of TGF-ß1 and contributes to the oncogenic conversion of TGF-ß1 function through activation of the ABCA1-Cav1-TßR signaling axis.