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The mitochondrion is a gatekeeper of apoptotic processes, and mediates drug resistance to several chemotherapy agents used to treat cancer. Neuroblastoma is a common solid cancer in young children with poor clinical outcomes following conventional chemotherapy. We sought druggable mitochondrial protein targets in neuroblastoma cells. Among mitochondria-associated gene targets, we found that high expression of the mitochondrial adenine nucleotide translocase 2 (SLC25A5/ANT2), was a strong predictor of poor neuroblastoma patient prognosis and contributed to a more malignant phenotype in pre-clinical models. Inhibiting this transporter with PENAO reduced cell viability in a panel of neuroblastoma cell lines in a TP53-status-dependant manner. We identified the histone deacetylase inhibitor, suberanilohydroxamic acid (SAHA), as the most effective drug in clinical use against mutant TP53 neuroblastoma cells. SAHA and PENAO synergistically reduced cell viability, and induced apoptosis, in neuroblastoma cells independent of TP53-status. The SAHA and PENAO drug combination significantly delayed tumour progression in pre-clinical neuroblastoma mouse models, suggesting that these clinically advanced inhibitors may be effective in treating the disease.

Introduction Clinical outcomes of implantable port catheters (IPCs) placed via alternative veins such as the external jugular and cervical collaterals have not been well established. This investigation evaluates the short- and long-term outcomes of IPCs inserted via alternate cervical veins (ACV) compared to traditionally inserted IPCs via the internal jugular vein (IJV). Materials and Methods A total of 24 patients who received an IPC between 2010 and 2020 via an ACV—defined as the external jugular vein, superficial cervical vein, or unnamed collateral veins—were identified. Based on power analysis, a matched control group of 72 patients who received IPCs via the IJV was identified. Non-inferiority analysis for port complications was performed between the two groups based on the selected non-inferiority margin of 20%. Secondary end points included complication-free survival and comparison of complications by the time at which they occurred. Results ACV access was non-inferior to traditional access for overall complications. Alternate access resulted in fewer complications than traditional access with an estimated reduction of − 7.0% [95% CI − 23.6%, 39.7%]. There was no significant difference in peri-procedural and post-procedural complications between the two groups. Complication-free survival was also equivalent between the two groups. Conclusion IPC placement via ACVs was non-inferior to IPCs placed via traditional access through the IJV. When abnormal pathology obviates the use of IJV access, other cervical veins may be considered prior to seeking alternate locations such as femoral, translumbar, inferior vena cava, and hepatic veins.

Histone deacetylase (HDAC) inhibitors are effective in MYCN-driven cancers, because of a unique need for HDAC recruitment by the MYCN oncogenic signal. However, HDAC inhibitors are much more effective in combination with other anti-cancer agents. To identify novel compounds which act synergistically with HDAC inhibitor, such as suberanoyl hydroxamic acid (SAHA), we performed a cell-based, high-throughput drug screen of 10,560 small molecule compounds from a drug-like diversity library and identified a small molecule compound (SE486-11) which synergistically enhanced the cytotoxic effects of SAHA. Effects of drug combinations on cell viability, proliferation, apoptosis and colony forming were assessed in a panel of neuroblastoma cell lines. Treatment with SAHA and SE486-11 increased MYCN ubiquitination and degradation, and markedly inhibited tumorigenesis in neuroblastoma xenografts, and, MYCN transgenic zebrafish and mice. The combination reduced ubiquitin-specific protease 5 (USP5) levels and increased unanchored polyubiquitin chains. Overexpression of USP5 rescued neuroblastoma cells from the cytopathic effects of the combination and reduced unanchored polyubiquitin, suggesting USP5 is a therapeutic target of the combination. SAHA and SE486-11 directly bound to USP5 and the drug combination exhibited a 100-fold higher binding to USP5 than individual drugs alone in microscale thermophoresis assays. MYCN bound to the USP5 promoter and induced USP5 gene expression suggesting that USP5 and MYCN expression created a forward positive feedback loop in neuroblastoma cells. Thus, USP5 acts as an oncogenic cofactor with MYCN in neuroblastoma and the novel combination of HDAC inhibitor with SE486-11 represents a novel therapeutic approach for the treatment of MYCN-driven neuroblastoma.

Clinical outcomes of implantable port catheters (IPCs) placed via alternative veins such as the external jugular and cervical collaterals have not been well established. This investigation evaluates the short- and long-term outcomes of IPCs inserted via alternate cervical veins (ACV) compared to traditionally inserted IPCs via the internal jugular vein (IJV).A total of 24 patients who received an IPC between 2010 and 2020 via an ACV-defined as the external jugular vein, superficial cervical vein, or unnamed collateral veins-were identified. Based on power analysis, a matched control group of 72 patients who received IPCs via the IJV was identified. Non-inferiority analysis for port complications was performed between the two groups based on the selected non-inferiority margin of 20%. Secondary end points included complication-free survival and comparison of complications by the time at which they occurred.ACV access was non-inferior to traditional access for overall complications. Alternate access resulted in fewer complications than traditional access with an estimated reduction of - 7.0% [95% CI - 23.6%, 39.7%]. There was no significant difference in peri-procedural and post-procedural complications between the two groups. Complication-free survival was also equivalent between the two groups.IPC placement via ACVs was non-inferior to IPCs placed via traditional access through the IJV. When abnormal pathology obviates the use of IJV access, other cervical veins may be considered prior to seeking alternate locations such as femoral, translumbar, inferior vena cava, and hepatic veins.

Investigations of anti-HIV-1 human hematopoietic stem/progenitor cell (HSPC)-based gene therapy have been performed by HIV-1 challenge after the engraftment of gene-modified HSPCs in humanized mouse models. However, the clinical application of gene therapy is to treat HIV-1-infected patients. Here, we developed a new method to investigate an anti-HIV-1 HSPC-based gene therapy in humanized mice previously infected with HIV-1. First, humanized mice were infected with HIV-1. When plasma viremia reached >107 copies/mL 3 weeks after HIV-1 infection, the mice were myeloablated with busulfan and transplanted with anti-HIV-1 gene-modified CD34+ HSPCs transduced with a lentiviral vector expressing two short hairpin RNAs (shRNAs) against CCR5 and HIV-1 long terminal repeat (LTR), along with human thymus tissue under the kidney capsule. Anti-HIV-1 vector-modified human CD34+ HSPCs successfully repopulated peripheral blood and lymphoid tissues in HIV-1 previously infected humanized mice. Anti-HIV-1 shRNA vector-modified CD4+ T lymphocytes showed selective advantage in HIV-1 previously infected humanized mice. This new method will be useful for investigations of anti-HIV-1 gene therapy when testing in a more clinically relevant experimental setting. Keywords: humanized mouse model, gene therapy for HIV/AIDS, HPSC-based gene therapy

Tripartite Motif-containing protein 16 (TRIM16) is a member of a large family of tripartite motif (TRIM) proteins, that has been implicated in the pathogenesis of multiple cancers. However, the mechanism by which TRIM16 acts as a tumour suppressor is currently unknown. We used the versatile yeast two-hybrid assay on a cDNA library from human testes, which has relative high TRIM16 expression, to identify potential TRIM16-binding proteins. We identified transactive response DNA-binding protein 43 (TDP43) as a novel TRIM16 binding protein. Co-immunoprecipitation assay demonstrated that TDP43 bound TRIM16 in neuroblastoma and breast cancer cells. Enforced over-expression of TRIM16 increased the protein half-life of TDP43, through the inhibition of the proteosomal degradation pathway. High levels of TRIM16 and TDP43 are associated with good prognosis in both human neuroblastoma and breast cancer tissues. Importantly, we found TDP43 expression was required for TRIM16-induced inhibition of neuroblastoma and breast cancer cell growth and the repressive effect of TRIM16 on cell cycle regulatory proteins, E2F1 and pRb. Taken together, our data suggest that TRIM16 and TDP43 are both good prognosis indicators; also we showed that TRIM16 inhibits cancer cell viability by a novel mechanism involving interaction and stabilisation of TDP43 with consequent effects on E2F1 and pRb proteins.

Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8(+) T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8(+) T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8(+) T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24(Gag) in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8(+) T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8(+) T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8(+) T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance.

The majority of patients diagnosed with neuroblastoma present with aggressive disease. Improved detection of neuroblastoma cancer cells following initial therapy may help in stratifying patient outcome and monitoring for relapse. To identify potential plasma biomarkers, we utilised a liquid chromatography tandem mass spectrometry-based proteomics approach to detect differentially-expressed proteins in serum from TH-MYCN mice. TH-MYCN mice carry multiple copies of the human MYCN oncogene in the germline and homozygous mice for the transgene develop neuroblastoma in a manner resembling the human disease. The abundance of plasma proteins was measured over the course of disease initiation and progression. A list of 86 candidate plasma biomarkers was generated. Pathway analysis identified significant association of these proteins with genes involved in the complement system. One candidate, complement C3 protein, was significantly enriched in the plasma of TH-MYCN+/+ mice at both 4 and 6 weeks of age, and was found to be elevated in a cohort of human neuroblastoma plasma samples, compared to healthy subjects. In conclusion, we have demonstrated the suitability of the TH-MYCN+/+ mouse model of neuroblastoma for identification of novel disease biomarkers in humans, and have identified Complement C3 as a candidate plasma biomarker for measuring disease state in neuroblastoma patients. Biological significance This study has utilised a unique murine model which develops neuroblastoma tumours that are biologically indistinguishable from human neuroblastoma. This animal model has effectively allowed the identification of plasma proteins which may serve as potential biomarkers of neuroblastoma. Furthermore, the label-free ion count quantitation technique which was used displays significant benefits as it is less labour intensive, feasible and accurate. We have been able to successfully validate this approach by confirming the differential abundance of two different plasma proteins. In addition, we have been able to confirm that the candidate biomarker Complement C3, is more abundant in the plasma of human neuroblastoma patient plasma samples when compared to healthy counterparts. Overall we have demonstrated that this approach can be potentially useful in the identification of biomarker candidates, and that further validation of the candidates may lead to the discovery of novel, clinically useful diagnostic tools in the detection of sub-clinical neuroblastoma. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved.

There is an urgent need for better therapeutic options for advanced melanoma patients, particularly those without the BRAFV600E/K mutation. In melanoma cells, loss of TRIM16 expression is a marker of cell migration and metastasis, while the BRAF inhibitor, vemurafenib, induces melanoma cell growth arrest in a TRIM16-dependent manner. Here we identify a novel small molecule compound which sensitized BRAF wild-type melanoma cells to vemurafenib. High throughput, cell-based, chemical library screening identified a compound (C012) which significantly reduced melanoma cell viability, with limited toxicity for normal human fibroblasts. When combined with the BRAFV600E/K inhibitor, vemurafenib, C012 synergistically increased vemurafenib potency in 5 BRAFWT and 4 out of 5 BRAFV600E human melanoma cell lines (Combination Index: CI < 1), and, dramatically reduced colony forming ability. In addition, this drug combination was significantly anti-tumorigenic in vivo in a melanoma xenograft mouse model. The combination of vemurafenib and C012 markedly increased expression of TRIM16 protein, and knockdown of TRIM16 significantly reduced the growth inhibitory effects of the vemurafenib and C012 combination. These findings suggest that the combination of C012 and vemurafenib may have therapeutic potential for the treatment of melanoma, and, that reactivation of TRIM16 may be an effective strategy for patients with this disease. Refereed/Peer-reviewed

Retinoid therapy is used for chemo-prevention in immuno-suppressed patients at high risk of developing skin cancer. The retinoid signalling molecule, tripartite motif protein 16 (TRIM16), is a regulator of keratinocyte differentiation and a tumour suppressor in retinoid-sensitive neuroblastoma. We sought to determine the role of TRIM16 in skin squamous cell carcinoma (SCC) pathogenesis. We have shown that TRIM16 expression was markedly reduced during the histological progression from normal skin to actinic keratosis and SCC. SCC cell lines exhibited lower cytoplasmic and nuclear TRIM16 expression compared with primary human keratinocyte (PHK) cells due to reduced TRIM16 protein stability. Overexpressed TRIM16 translocated to the nucleus, inducing growth arrest and cell differentiation. In SCC cells, TRIM16 bound to and down regulated nuclear E2F1, this is required for cell replication. Retinoid treatment increased nuclear TRIM16 expression in retinoid-sensitive PHK cells, but not in retinoid-resistant SCC cells. Overexpression of TRIM16 reduced SCC cell migration, which required the C-terminal RET finger protein (RFP)-like domain of TRIM16. The mesenchymal intermediate filament protein, vimentin, was directly bound and down-regulated by TRIM16 and was required for TRIM16-reduced cell migration. Taken together, our data suggest that loss of TRIM16 expression plays an important role in the development of cutaneous SCC and is a determinant of retinoid sensitivity. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The family of tripartite-motif (TRIM) proteins are involved in diverse cellular processes, but are often characterized by critical protein-protein interactions necessary for their function. TRIM16 is induced in different cancer types, when the cancer cell is forced to proceed down a differentiation pathway. We have identified TRIM16 as a DNA-binding protein with histone acetylase activity, which is required for the retinoic acid receptor β(2) transcriptional response in retinoid-treated cancer cells. In this study, we show that overexpressed TRIM16 reduced neuroblastoma cell growth, enhanced retinoid-induced differentiation and reduced tumourigenicity in vivo. TRIM16 was only expressed in the differentiated ganglion cell component of primary human neuroblastoma tumour tissues. TRIM16 bound directly to cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells. TRIM16 reduced cell motility and this required downregulation of vimentin. Retinoid treatment and enforced overexpression caused TRIM16 to translocate to the nucleus, and bind to and downregulate nuclear E2F1, required for cell replication. This study, for the first time, demonstrates that TRIM16 acts as a tumour suppressor, affecting neuritic differentiation, cell migration and replication through interactions with cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells.

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, FAcilitates Chromatin Transcription (FACT), 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.

Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT) mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC) transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA). However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR). Here, we report that human CD4+T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4+T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4+T-cells ex vivo. Furthermore, levels of gene-marked CD4+T-cells in peripheral blood increased despite systemic infection with either CXCR4- or CCR5- tropic HIV-1 in vivo. These results demonstrate that transplantation of HSPCs engineered with our combination shRNA vector may be a potential therapy against HIV disease. © 2012 Ringpis et al.

Neuroblastoma is a childhood cancer of the sympathetic nervous system. Approximately 20% of patients present with an aggressive metastatic disease characterized by amplification of the MYCN locus. However, an additional 30% of patients suffer from an equally aggressive disease, yet there are no unified genetic markers available. To identify potential biomarkers of poor prognosis in neuroblastoma, we conducted unsupervised hierarchical clustering on 649 primary neuroblastoma tumors based on a 51 gene MYC core target signature. This demonstrated strong activation within the MYCN amplified cohort as expected, but surprisingly there was a large subset of MYCN non-amplified patients that exhibited high MYC signaling. High expression of this MYC signature proved to be strongly predictive of poor overall survival, in both the full cohort (Hazard ratio (HR): 47.6, p= 2.7x10-35, n=649) and in MYCN non-amplified patients (HR=32.8, p=1.3x10-13, n=554), suggesting that neuroblastoma can be driven by MYC signaling in the absence of MYCN amplification. To evaluate potential therapeutic targets within this MYC signature, we conducted cox regression analysis on the 51 individual members and identified that SPT16 was a particularly potent predictor of poor outcome in these 649 neuroblastoma patients (HR: 7.2, p=2.7x10-17). SPT16 is a subunit of the FACT histone chaperone complex, which together with its heterodimer partner SSRP1, has previously been shown to function in dynamic regulation of chromatin to drive transcription, DNA replication and DNA repair. Similar to SPT16, high SSRP1 expression was a strong predictor of poor prognosis in 649 neuroblastoma patients (HR: 12.5, p=5.812x10-26) implicating FACT as a mediator of MYC driven neuroblastoma. Next we investigated the biological relationship between FACT and MYCN in neuroblastoma. siRNA experiments showed that FACT and MYCN participate in a positive feedback transcriptional loop that is essential for maintaining mutual high expression. Immunohistochemistry in sympathetic ganglia of pre-tumor TH-MYCN mouse model of neuroblastoma identified that FACT was highly expressed at tumor initiation and chemical inhibition of FACT by CBL0137 markedly reduced tumor initiation and subsequent tumorigenesis in vivo. Next we tested CBL0137 against established neuroblastoma in TH-MYCN mice. CBL0137 was highly cytotoxic to tumors either as a single agent or by synergistically promoting clinically used chemotherapeutics. This was accompanied by marked MYCN depletion and prolonged tumor regression in a majority of recipient mice. Pulsed-field electrophoresis showed that CBL0137 impaired FACT dependent DNA damage repair in the presence of genotoxic chemotherapy, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells. Together our data define FACT as a mediator of neuroblastoma tumorigenesis and MYCN-related treatment target. Given the current poor survival rates of children with high-risk neuroblastoma, our findings provide support for the clinical development of CBL0137 as a novel treatment approach for this refractory malignancy. Citation Format: Daniel R. Carter, Jayne Murray, Belamy B. Cheung, Heyam Kalla, Laura Gamble, Joanna Tsang, Selina Sutton, Jessica Koach, Sarah Syed, Andrew Gifford, Natalia Issaeva, Asel Biktasova, Bernard Atmadibrata, Yuting Sun, Nicolas Sokolowski, Dora Ling, Patrick Y. Kim, Hannah Webber, Ashleigh Clark, Michelle Ruhle, Bing Liu, André Oberthuer, Matthias Fischer, Jennifer Byrne, Federica Saletta, Andrei Purmal, David Ziegler, Tao Liu, Katerina V. Gurova, Andrei V. Gudkov, Murray D. Norris, Michelle Haber, Glenn M. Marshall. MYCN and is a therapeutic target in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR09.

The tripartite motif (TRIM) family of proteins are involved in a diverse range of cellular processes ranging from innate immunity, oncogenesis as well as tumor suppression. A majority of these reported functions occur through protein-protein interactions. Previously, we have found a strong correlation between loss of TRIM16 expression and disease progression in three human cancers: neuroblastoma, squamous cell carcinoma and melanoma. Moreover, our studies have shown that TRIM16 acts as a tumor suppressor in these cancers through effects on cell cycle and migration. In order to better understand the mechanism of TRIM16 in cancer cell growth inhibition, we have identified a novel binding partner of TRIM16 and investigated the functional outcome of the protein-protein interaction. Using the yeast two-hybrid screening tool, we identified transactive response DNA-binding protein 43 (TDP43) as a novel TRIM16 binding protein. TDP43 has been described as a RNA/DNA binding protein with a putative role in HIV transcription and reports also suggest that this protein plays a role in a wide range of neurodegenerative diseases. Through co-immunoprecipitation studies, we have demonstrated that TDP43 directly binds to TRIM16 in human BE(2)-C neuroblastoma and MCF7 breast cancer cells. Enforced over-expression of TRIM16 increased the protein half-life of TDP43. We have also shown that TDP43 expression was required for TRIM16 induced inhibition of neuroblastoma and breast cancer cell growth. Moreover, we have shown that two cell cycle regulatory proteins, E2F1 and pRb, were regulated by TRIM16 and TDP43. Taken together, our studies suggest that the novel mechanism by which TRIM16 inhibits cancer cell growth is through the direct interaction and up-regulation of TDP43 protein expression and with consequent effects on E2F1 and phos-Rb. Citation Format: Patrick Y. Kim, Owen Tan, Toby Trahair, Tao Liu, Glenn M. Marshall, Belamy B. Cheung. TRIM16 inhibits cell growth through direct interaction and modulation of TDP43 protein stability in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1403. doi:10.1158/1538-7445.AM2014-1403

TRIM16 exhibits tumour suppressor functions by interacting with cytoplasmic vimentin and nuclear E2F1 proteins in neuroblastoma and squamous cell carcinoma cells, reducing cell migration and replication. Reduced TRIM16 expression in a range of human primary malignant tissues correlates with increased malignant potential. TRIM16 also induces apoptosis in breast and lung cancer cells, by unknown mechanisms. Here we show that overexpression of TRIM16 induces apoptosis in human breast cancer (MCF7) and neuroblastoma (BE(2)-C) cells, but not in non-malignant HEK293 cells. TRIM16 increased procaspase-2 protein levels in MCF7 and induced caspase-2 activity in both MCF7 and BE(2)-C cells. We show that TRIM16 and caspase-2 proteins directly interact in both MCF7 and BE(2)-C cells and co-localise in MCF7 cells. Most importantly, the induction of caspase-2 activity is required for TRIM16 to initiate apoptosis. Our data suggest a novel mechanism by which TRIM16 can promote apoptosis by directly modulating caspase-2 activity.