SUMMARY Using sequential gene expression profiling (GEP) samples, we defined a major functional group related to drug resistance that contains chromosomal instability (CIN) genes. One CIN gene in particular, NEK2, was highly correlated with drug resistance, rapid relapse, and poor outcome in multiple cancers. Over-expressing NEK2 in cancer cells resulted in enhanced CIN, cell proliferation and drug resistance, while targeting NEK2 by NEK2 shRNA overcame cancer cell drug resistance and induced apoptosis in vitro and in a xenograft myeloma mouse model. High expression of NEK2 induced drug resistance mainly through activation of the efflux pumps. Thus, NEK2 represents a strong predictor for drug resistance and poor prognosis in cancer and could be an important target for cancer therapy.

Aims-FoxO4 is a member of the forkhead box transcription factor O (FoxO) subfamily. FoxO proteins are involved in diverse biological processes. In this study, we examine the role of FoxO4 in intestinal mucosal immunity and inflammatory bowel disease (IBD).Methods-Foxo4-null mice were subjected to trinitrobenzene sulfonic acid (TNBS)-treatment. Microarray analysis and quantitative RT-PCR were used to identify the cytokine transcripts that were altered by Foxo4-deletion. The effects of Foxo4-deficiency on the intestinal epithelial permeability and levels of tight junction proteins were examined by permeable fluorescent dye and western blot. The molecular and cellular mechanism(s) by which FoxO4 regulates the mucosal immunity were explored through immunological and biochemical analyses. The expression level of FoxO4 in intestinal epithelial cells of patients with IBD was examined using immunohistochemistry.Results-Foxo4-null mice were more susceptible to TNBS injury induced colitis. The chemokine CCL5 is significantly up-regulated in the colonic epithelial cells of Foxo4-null mice, with increased recruitment of CD4+ intraepithelial T cells and up-regulation of cytokines INFγ and TNFα in the colon. Foxo4-deficiency also resulted in an increase in intestinal epithelial permeability and downregulation of the tight junction proteins ZO-1 and claudin-1. Mechanistically, FoxO4 inhibited the transcriptional activity of NF-κB and Foxo4-deficiency is associated with increased NF-κB activity in vivo. FoxO4 transcription is transiently repressed in response to TNBS treatment and in patients with IBD. *To whom correspondence should be addressed:, Ph: 214 648-1485, Fx: 214 648-1450, E-mail: Zhi-Ping.Liu@utsouthwestern.edu. Author contribution: Wen Zhou, Qian Cao, and Qing-Jun Zhang-acquisition of data, analysis and interpretation of data; Yan Penganalysis and interpretation of data; Diego H. Castrillon and Ronald A. DePinho-material support, drafting of the manuscript; Zhi-Ping Liu-study concept and design, drafting of the manuscript, analysis and interpretation of data, and obtained funding.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conclusion-These results indicate that FoxO4 is an endogenous inhibitor of NF-κB and identify a novel function of FoxO4 in the regulation of NF-κB mediated mucosal immunity.

BackgroundNasopharyngeal carcinoma (NPC) is a common malignant tumor in southern China and Southeast Asia, but its molecular mechanisms of pathogenesis are poorly understood. Our previous work has demonstrated that BCAT1 mRNA is over expressed in NPC and knocking down its expression in 5-8F NPC cell line can potently inhibit cell cycle progression and cell proliferation. However, the mechanism of BCAT1 up-regulation and its functional role in NPC development remain to be elucidated yet.MethodsImmunohistochemistry (IHC) method was utilized to detect the expression of BCAT1 protein in NPC at different pathological stages. The roles of gene mutation, DNA amplification and transcription factor c-Myc in regulating BCAT1 expression were analyzed using PCR-sequencing, quantitative polymerase chain reaction (qPCR), IHC, ChIP and luciferase reporter system, respectively. The functions of BCAT1 in colony formation, cell migration and invasion properties were evaluated by RNA interference (RNAi).ResultsThe positive rates of BCAT1 protein expression in normal epithelia, low-to-moderate grade atypical hyperplasia tissues, high-grade atypical hyperplasia tissues and NPC tissues were 23.6% (17/72), 75% (18/24 ), 88.9% (8/9) and 88.8% (71/80), respectively. Only one SNP site in exon1 was detected, and 42.4% (12/28) of the NPC tissues displayed the amplification of microsatellite loci in BCAT1. C-Myc could directly bind to the c-Myc binding site in promoter region of BCAT1 and up-regulate its expression. The mRNA and protein of c-Myc and BCAT1 were co-expressed in 53.6% (15/28) and 59.1% (13/22) of NPC tissues, respectively, and BCAT1 mRNA expression was also down-regulated in c-Myc knockdown cell lines. In addition, BCAT1 knockdown cells demonstrated reduced proliferation and decreased cell migration and invasion abilities.ConclusionsOur study indicates that gene amplification and c-Myc up-regulation are responsible for BCAT1 overexpression in primary NPC, and overexpression of BCAT1 induces cell proliferation, migration and invasion. The results suggest that BCAT1 may be a novel molecular target for the diagnosis and treatment of NPC.

Key Points• RARa2 activates Wnt and hedgehog pathways in maintaining myeloma stem cell features and drug resistance.We previously demonstrated that RARa2 expression is increased in CD138 selected plasma cells of relapsed multiple myelomas (MMs), and increased expression was linked to poor prognosis in newly diagnosed MM patients. In the present study, we demonstrate that increased RARa2 confers myeloma stem cell features. Higher expression of RARa2 was identified in the multiple myeloma stem cell (MMSC) fraction. Overexpression of RARa2 in bulk MM cell lines resulted in: 1) increased drug resistance; 2) increased clonogenic potential; 3) activation of both Wnt and Hedgehog (Hh) pathways; 4) increased side population and aldehyde dehydrogenase levels; and 5) increased expression of embryonic stem cell genes. The opposite effects were seen with RARa2 knockdown. We demonstrate that RARa2 induces drug resistance by activating the drug efflux pump gene ABCC3 and anti-apoptotic Bcl-2 family members. Inhibition of Wnt signaling or ABCC3 function could overcome drug resistance in RARa2 overexpressing MM cells. We also showed that in the 5TGM1 mouse model, targeting of the Wnt and Hh pathways using CAY10404, cyclopamine, or itraconazole significantly reduced the myeloma tumor burden and increased survival. Targeting RARa2 or its downstream signaling pathways provides a potential strategy to eliminate

NEK2 is associated with drug resistance in multiple cancers. Our previous studies indicated that high NEK2 confers inferior survival in multiple myeloma (MM); thus, a better understanding of the mechanisms by which NEK2 induces drug resistance in MM is required. In this study, we discovered that NEK2 enhances MM cell autophagy, and a combination of autophagy inhibitor chloroquine (CQ) and chemotherapeutic bortezomib (BTZ) significantly prevents NEK2‐induced drug resistance in MM cells. Interestingly, NEK2 was found to bind and stabilize Beclin‐1 protein but did not affect its mRNA expression and phosphorylation. Moreover, autophagy enhanced by NEK2 was significantly prevented by knockdown of Beclin‐1 in MM cells, suggesting that Beclin‐1 mediates NEK2‐induced autophagy. Further studies demonstrated that Beclin‐1 ubiquitination is decreased through NEK2 interaction with USP7. Importantly, knockdown of Beclin‐1 sensitized NEK2‐overexpressing MM cells to BTZ in vitro and in vivo. In conclusion, we identify a novel mechanism whereby autophagy is activated by the complex of NEK2/USP7/Beclin‐1 in MM cells. Targeting the autophagy signaling pathway may provide a promising therapeutic strategy to overcome NEK2‐induced drug resistance in MM.

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Human leucine zipper transcription factor-like 1 (LZTFL1) is a novel gene with unknown biological functions. It is located in the chromosome region 3p21.3, a hotspot for tumor suppressor genes. To understand the biological functions of LZTFL1, we surveyed the expression level of LZTFL1 in tumor and normal samples in tissue microarrays and a clinical archive of 84 gastric cancer specimens using immunohistochemistry. We found that LZTFL1 is expressed highly in the epithelial cells of normal tissues and is significantly downregulated in the corresponding tumor samples. The expression level of LZTFL1 correlated significantly with the survival outcomes of the patients and had significant inverse correlation with tumor metastasis. Overexpression of LZTFL1 in tumor cells inhibited anchorage-independent cell growth and cell migration in vitro and repressed tumor growth in vivo. Furthermore, we show that LZTFL1 expression is upregulated on epithelial cell differentiation and is graded along the crypt-villus axis of the intestine, with weakest expression level in the proliferative zone of the crypt and highest expression level at the apex of the differentiation zone in the villus. Expression of LZTFL1 overlaps with that of E-cadherin at the plasma membrane. Our results indicate that LZTFL1 is a tumor suppressor and that loss of LZTFL1 expression has significant clinical outcomes. LZTFL1 expression may serve as an independent prognostic marker for survival outcome of gastric cancer patients. We propose that LZTFL1 may inhibit tumorigenesis by stabilizing E-cadherin-mediated adherens junction formation and promoting epithelial cell differentiation. Cancer Res; 70(7); 2942-50. ©2010 AACR.

SummaryThe serine synthesis pathway (SSP) is active in multiple cancers. Previous study has shown that bortezomib (BTZ) resistance is associated with an increase in the SSP in multiple myeloma (MM) cells; however, the underlying mechanisms of SSP‐induced BTZ resistance remain unclear. In this study, we found that phosphoglycerate dehydrogenase (PHGDH), the first rate‐limiting enzyme in the SSP, was significantly elevated in CD138+ cells derived from patients with relapsed MM. Moreover, high PHGDH conferred inferior survival in MM. We also found that overexpression of PHDGH in MM cells led to increased cell growth, tumour formation, and resistance to BTZ in vitro and in vivo, while inhibition of PHGDH by short hairpin RNA or NCT‐503, a specific inhibitor of PHGDH, inhibited cell growth and BTZ resistance in MM cells. Subsequent mechanistic studies demonstrated PHGDH decreased reactive oxygen species (ROS) through increasing reduced glutathione (GSH) synthesis, thereby promoting cell growth and BTZ resistance in MM cells. Furthermore, adding GSH to PHGDH silenced MM cells reversed S phase arrest and BTZ‐induced cell death. These findings support a mechanism in which PHGDH promotes proliferation and BTZ resistance through increasing GSH synthesis in MM cells. Therefore, targeting PHGDH is a promising strategy for MM therapy.