Zhaoxia Qu

Hsp90 regulates processing of NF-κB2 p100 involving protection of NF-κB-inducing kinase (NIK) from autophagy-mediated degradation

NF-κB-inducing kinase (NIK) is required for NF-κB activation based on the processing of NF-κB2 p100. Here we report a novel mechanism of NIK regulation involving the chaperone 90 kDa heat shock protein (Hsp90) and autophagy. Functional inhibition of Hsp90 by the anti-tumor agent geldanamycin (GA) efficiently disrupts its interaction with NIK, resulting in NIK degradation and subsequent blockage of p100 processing. Surprisingly, GA-induced NIK degradation is mediated by autophagy, but largely independent of the ubiquitin-proteasome system. Hsp90 seems to be specifically involved in the folding/stabilization of NIK protein, because GA inhibition does not affect NIK mRNA transcription and translation. Furthermore, Hsp90 is not required for NIK-mediated recruitment of the α subunit of IκB kinase to p100, a key step in induction of p100 processing. These findings define an alternative mechanism for Hsp90 client degradation and identify a novel function of autophagy in NF-κB regulation. These findings also suggest a new therapeutic strategy for diseases associated with p100 processing.

Stabilization of Basally Translated NF-κB-inducing Kinase (NIK) Protein Functions as a Molecular Switch of Processing of NF-κB2 p100

The non-canonical pathway based on processing of NF-κB2 precursor protein p100 to generate p52 plays a critical role in controlling B cell function and lymphoid organogenesis. Activation of this unique pathway by extracellular stimuli requires NF-κB-inducing kinase (NIK) and de novo protein synthesis. However, how NIK is regulated is largely unknown. Here, we systematically analyzed NIK expression at different levels in the presence or absence of different NF-κB stimuli. We found that NIK mRNA is relatively abundant and undergoes constitutive protein synthesis in resting B cells. However, NIK protein is undetectable. Interestingly, protein expression of NIK is steadily induced by B cell-activating factor or CD40 ligand, two major physiological inducers of p100 processing, but not by mitogen phorbol 12-myristate 13-acetate/ionomycin or cytokine tumor necrosis factor α, two well known inducers of the canonical NF-κB signaling. Remarkably, both B cell-activating factor and CD40 ligand do not significantly induce expression of NIK at translational or transcriptional level but rather rescue the basally translated NIK protein from undergoing degradation. Furthermore, overexpressed or purified NIK protein triggers p100 processing in the presence of protein synthesis inhibitor. Taken together, these studies define one important mechanism of NIK regulation and the central role of NIK stabilization in the induction of p100 processing. These studies also provide the first evidence explaining why activation of the non-canonical NF-κB signaling is delayed and can be inhibited by protein synthesis inhibitor as well as why most classical NF-κB stimuli, including mitogens and tumor necrosis factor α, fail to induce p100 processing.

PDLIM2 suppresses human T-cell leukemia virus type I Tax-mediated tumorigenesis by targeting Tax into the nuclear matrix for proteasomal degradation

The mechanisms by which the human T-cell leukemia virus type I (HTLV-I) Tax oncoprotein deregulates cellular signaling for oncogenesis have been extensively studied, but how Tax itself is regulated remains largely unknown. Here we report that Tax was negatively regulated by PDLIM2, which promoted Tax K48-linked polyubiquitination. In addition, PDLIM2 recruited Tax from its functional sites into the nuclear matrix where the polyubiquitinated Tax was degraded by the proteasome. Consistently, PDLIM2 suppressed Tax-mediated signaling activation, cell transformation, and oncogenesis both in vitro and in animal. Notably, PDLIM2 expression was down-regulated in HTLV-I-transformed T cells, and PDLIM2 reconstitution reversed the tumorigenicity of the malignant cells. These studies indicate that the counterbalance between HTLV-I/Tax and PDLIM2 may determine the outcome of HTLV-I infection. These studies also suggest a potential therapeutic strategy for cancers and other diseases associated with HTLV-I infection and/or PDLIM2 deregulation.

RUNX3 acts as a tumor suppressor in breast cancer by targeting estrogen receptor α

Transcription factor RUNX3 is inactivated in a number of malignancies, including breast cancer, and is suggested to function as a tumor suppressor. How RUNX3 functions as a tumor suppressor in breast cancer remains undefined. Here, we show that about 20% of female Runx3+/− mice spontaneously developed ductal carcinoma at an average age of 14.5 months. Additionally, RUNX3 inhibits the estrogen-dependent proliferation and transformation potential of ERα-positive MCF-7 breast cancer cells in liquid culture and in soft agar and suppresses the tumorigenicity of MCF-7 cells in severe combined immunodeficiency mice. Furthermore, RUNX3 inhibits ERα-dependent transactivation by reducing the stability of ERα. Consistent with its ability to regulate the levels of ERα, expression of RUNX3 inversely correlates with the expression of ERα in breast cancer cell lines, human breast cancer tissues and Runx3+/− mouse mammary tumors. By destabilizing ERα, RUNX3 acts as a novel tumor suppressor in breast cancer.

The tumor suppressor gene WWOX links the canonical and noncanonical NF-κB pathways in HTLV-I Tax-mediated tumorigenesis

Both the canonical and noncanonical nuclear factor κB (NF-κB) pathways have been linked to tumorigenesis. However, it remains unknown whether and how the 2 signaling pathways cooperate during tumorigenesis. We report that inhibition of the noncanonical NF-κB pathway significantly delays tumorigenesis mediated by the viral oncoprotein Tax. One function of noncanonical NF-κB activation was to repress expression of the WWOX tumor suppressor gene. Notably, WWOX specifically inhibited Tax-induced activation of the canonical, but not the noncanonical NF-κB pathway. Mechanistic studies indicated that WWOX blocked Tax-induced inhibitors of κB kinaseα (IKKα) recruitment to RelA and subsequent RelA phosphorylation at S536. In contrast, WWOX Y33R, a mutant unable to block the IKKα recruitment and RelA phosphorylation, lost the ability to inhibit Tax-mediated tumorigenesis. These data provide one important mechanism by which Tax coordinates the 2 NF-κB pathways for tumorigenesis. These data also suggest a novel role of WWOX in NF-κB regulation and viral tumorigenesis.

Epigenetic Repression of PDZ-LIM Domain-containing Protein 2 IMPLICATIONS FOR THE BIOLOGY AND TREATMENT OF BREAST CANCER

The NF-κB transcription factor plays a pivotal role in breast cancer progression and therapy resistance. However, the mechanisms by which the tightly regulated NF-κB becomes constitutively activated during breast cancer pathogenesis remain obscure. Here, we report that PDZ-LIM domain-containing protein 2 (PDLIM2), an essential terminator of NF-κB activation, is repressed in both estrogen receptor-positive and estrogen receptor-negative breast cancer cells, suggesting one important mechanism for the constitutive activation of NF-κB. Indeed, PDLIM2 reexpression inhibited constitutive NF-κB activation and expression of NF-κB-targeted genes in those breast cancer cells. Importantly, PDLIM2, but not its mutants defective in NF-κB termination, could suppress in vitro anchorage-independent growth and in vivo tumor formation of those malignant breast cells. In addition, we have shown that PDLIM2 repression involves promoter methylation. Accordingly, treatment of the breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine reverses the methylation of the PDLIM2 promoter, restored PDLIM2 expression, and suppressed tumorigenicities of human breast cancer cells both in vitro and in vivo. These studies thus provide important mechanistic insights into breast cancer pathogenesis. These studies also suggest a tumor suppression function of PDLIM2 and a therapeutic strategy for breast cancer.

Targeting Autophagic Regulation of NF-kappaB in HTLV-I Transformed Cells by Geldanamycin: Implications for Therapeutic Interventions

The IκappaB kinase (IKK)/NF-κappaB signaling pathway plays an essential role in the development and survival of many types of cancers including adult T-cell leukemia (ATL) caused by the human T-cell leukemia virus type I (HTLV-I) infection. Accordingly, targeting NF-κappaB provides an attractive strategy for cancer therapy. We recently found that specific inhibition of Hsp90 by geldanamycin (GA) results in autophagic degradation of IKK and NF-κappaB-inducing kinase (NIK), an upstream kinase of IKK, and inactivation of NF-κappaB in various cell lines. Here, we further report that GA inhibition of Hsp90 also led to IKK autophagic degradation and NF-κappaB inhibition in both HTLV-transformed T cells and ATL-derived cell lines. Importantly, GA treatment led to efficient apoptosis of these malignant cells, whereas inhibition of autophagic degradation of IKK significantly ameliorated the cytotoxic effect of GA. These findings thus not only provide mechanistic insights into the tumor suppression function of autophagy and the anti-tumor activity of GA, but also suggest an immediate therapeutic strategy for ATL and other diseases associated with NF-κappaB activation by targeting autophagic degradation of the central NF-kappaB activating kinases. Addendum to: Hsp90 Inhibition Results in Autophagy-Mediated Proteasome-Independent Degradation of IκappaB Kinase (IKK) G. Qing, P. Yan and G. Xiao Cell Res 2006; 16:895-901 and Hsp90 Regulates Processing of NF-κappaB2 p100 Involving Protection of NF-κappaB-Inducing Kinase (NIK) from Autophagy-Mediated Degradation G. Qing, P. Yan, Z. Qu, H. Liu and G. Xiao Cell Res 2007; 17:520-30

Endoproteolytic processing of C-terminally truncated NF-κB2 precursors at κB-containing promoters

The C-terminal, partially truncated forms of the NF-κB2/p52 precursor p100, p100ΔCs, manifest constitutive processing and oncogenic ability, although the responsible mechanisms remain unknown. Here, we report that p100ΔCs are specifically processed in association with binding to promoter DNA-containing κB sites. In the nucleus, p100ΔCs bind to the κB promoter DNA and subsequently recruit the proteasome to form a stable proteasome/p100ΔC/DNA complex, which mediates the processing of p100ΔCs. Notably, the processing at the κB promoter is initiated by a proteasome-mediated endoproteolytic cleavage at amino acid D415 of p100ΔCs, and the processed p52, but not the precursors themselves, is oncogenic by up-regulating a subset of target genes. Our studies demonstrate a different mechanism of p100 processing and also present evidence showing that the proteasome modulates the action of transcription factors at promoter regions through endoproteolysis.

Differential roles of STAT3 in the initiation and growth of lung cancer.

Signal transducer and activator of transcription 3 (STAT3) is linked to multiple cancers, including pulmonary adenocarcinoma. However, the role of STAT3 in lung cancer pathogenesis has not been determined. Using lung epithelial-specific inducible knockout strategies, we demonstrate that STAT3 has contrasting roles in the initiation and growth of both chemically and genetically induced lung cancers. Selective deletion of lung epithelial STAT3 in mice before cancer induction by the smoke carcinogen, urethane, resulted in increased lung tissue damage and inflammation, K-Ras oncogenic mutations and tumorigenesis. Deletion of lung epithelial STAT3 after establishment of lung cancer inhibited cancer cell proliferation. Simultaneous deletion of STAT3 and expression of oncogenic K-Ras in mouse lung elevated pulmonary injury, inflammation and tumorigenesis, but reduced tumor growth. These studies indicate that STAT3 prevents lung cancer initiation by maintaining pulmonary homeostasis under oncogenic stress, whereas it facilitates lung cancer progression by promoting cancer cell growth. These studies also provide a mechanistic basis for targeting STAT3 to lung cancer therapy.

Interleukin-6 Prevents the Initiation but Enhances the Progression of Lung Cancer

Recent studies suggest that high expression of the proinflammatory cytokine IL6 is associated with poor survival of lung cancer patients. Accordingly, IL6 has been a target of great interest for lung cancer therapy. However, the role of IL6 in lung cancer has not been determined yet. Here, we demonstrate that IL6 plays opposite roles in the initiation and growth of lung cancer in a mouse model of lung cancer induced by the K-Ras oncogene. We find that compared with wild-type mice, IL6-deficient mice developed much more lung tumors after an activating mutant of K-Ras was induced in the lungs. However, lung tumors developed in IL6-deficient mice were significantly smaller. Notably, both the lung tumor-suppressing and -promoting functions of IL6 involve its ability in activating the transcription factor STAT3. IL6/STAT3 signaling suppressed lung cancer initiation through maintaining lung homeostasis, regulating lung macrophages, and activating cytotoxic CD8 T cells under K-Ras oncogenic stress, whereas it promoted lung cancer cell growth through inducing the cell proliferation regulator cyclin D1. These studies reveal a previously unexplored role of IL6/STAT3 signaling in maintaining lung homeostasis and suppressing lung cancer induction. These studies also significantly improve our understanding of lung cancer and provide a molecular basis for designing IL6/STAT3-targeted therapies for this deadliest human cancer.