Swathi V. Iyer

DNAJA1 controls the fate of misfolded mutant p53 through the mevalonate pathway

Stabilization of mutant p53 (mutp53) in tumours greatly contributes to malignant progression. However, little is known about the underlying mechanisms and therapeutic approaches to destabilize mutp53. Here, through high-throughput screening we identify statins, cholesterol-lowering drugs, as degradation inducers for conformational or misfolded p53 mutants with minimal effects on wild-type p53 (wtp53) and DNA contact mutants. Statins preferentially suppress mutp53-expressing cancer cell growth. Specific reduction of mevalonate-5-phosphate by statins or mevalonate kinase knockdown induces CHIP ubiquitin ligase-mediated nuclear export, ubiquitylation, and degradation of mutp53 by impairing interaction of mutp53 with DNAJA1, a Hsp40 family member. Knockdown of DNAJA1 also induces CHIP-mediated mutp53 degradation, while its overexpression antagonizes statin-induced mutp53 degradation. Our study reveals that DNAJA1 controls the fate of misfolded mutp53, provides insights into potential strategies to deplete mutp53 through the mevalonate pathway–DNAJA1 axis, and highlights the significance of p53 status in impacting statins’ efficacy on cancer therapy.

MTBP suppresses cell migration and filopodia formation by inhibiting ACTN4.

Murine double minute (MDM2) binding protein (MTBP) has been implicated in cancer progression. Here, we demonstrate one mechanism by which MTBP inhibits cancer metastasis. Overexpression of MTBP in human osteosarcoma cell lines lacking wild-type p53 did not alter primary tumor growth in mice, but significantly inhibited metastases. MTBP downregulation increased the migratory potential of MDM2−/−p53−/− mouse embryonic fibroblasts, suggesting that MTBP inhibited cell migration independently of the Mdm2–p53 pathway. Co-immunoprecipitation and mass spectrometric analysis identified alpha-actinin-4 (ACTN4) as an MTBP-interacting protein. Endogenous MTBP interacted with and partially colocalized with ACTN4. MTBP overexpression inhibited cell migration and filopodia formation mediated by ACTN4. Increased cell migration by MTBP downregulation was inhibited by concomitant downregulation of ACTN4. MTBP also inhibited ACTN4-mediated F-actin bundling. We furthermore demonstrated that nuclear localization of MTBP was dispensable for inhibiting ACTN4-mediated cell migration and filopodia formation. Thus, MTBP suppresses cell migration, at least partially, by inhibiting ACTN4 function. Our study not only provides a mechanism of metastasis suppression by MTBP, but also suggests MTBP as a potential biomarker for cancer progression.

Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities

Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies.

Genome-wide RNAi screening identifies TMIGD3 isoform1 as a suppressor of NF-κB and osteosarcoma progression.

The ability of cancer cells to survive and grow in anchorage- and serum-independent conditions is well correlated with their aggressiveness. Here, using a human whole-genome shRNA library, we identify TMIGD3 isoform1 (i1) as a factor that suppresses this ability in osteosarcoma (OS) cells, mainly by inhibiting NF-κB activity. Knockdown of TMIGD3 increases proliferation, tumour formation and metastasis of OS cells. Overexpression of TMIGD3 isoform1 (i1), but not isoform3 (i3) which shares a common C-terminal region, suppresses these malignant properties. Adenosine A3 receptor (A3AR) having an identical N-terminal region shows similar biological profiles to TMIGD3 i1. Protein expression of TMIGD3 and A3AR is lower in human OS tissues than normal tissues. Mechanistically, TMIGD3 i1 and A3AR commonly inhibit the PKA−Akt−NF-κB axis. However, TMIGD3 i1 only partially rescues phenotypes induced by A3AR knockdown, suggesting the presence of distinct pathways. Our findings reveal an unappreciated role for TMIGD3 i1 as a suppressor of NF-κB activity and OS progression.

Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma

Objectives The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318–1, a murine osteosarcoma cell line. Methods The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP) and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency. Key findings Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent. Conclusions This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.

An improved intrafemoral injection with minimized leakage as an orthotopic mouse model of osteosarcoma

Osteosarcoma, the most common type of primary bone cancer, is the second highest cause of cancer-related death in pediatric patients. To understand the mechanisms behind osteosarcoma progression and to discover novel therapeutic strategies for this disease, a reliable and appropriate mouse model is essential. For this purpose, osteosarcoma cells need to be injected into the bone marrow. Previously, the intratibial and intrafemoral injection methods were reported; however, the major drawback of these methods is the potential leakage of tumor cells from the injection site during or after these procedures. To overcome this, we have established an improved method to minimize leakage in an orthotopic mouse model of osteosarcoma. By taking advantage of the anatomical benefits of the femur with less bowing and larger medullary cavity than those of the tibia, osteosarcoma cells are injected directly into the femoral cavity following reaming of its intramedullary space. To prevent potential leakage of tumor cells during and after the surgery, the injection site is sealed with bone wax. This method requires a minor surgery of approximately 15min under anesthesia. Our established orthotopic osteosarcoma model could serve as a valuable and reliable tool for examining progression of various types of bone tumors.

Suppressive roles of A3AR and TMIGD3 i1 in osteosarcoma malignancy

Osteosarcoma (OS) is themost commonmalignant tumor in bone affecting children and adolescents. Since majority of the patients manifest micrometastases at the time of diagnosis, chemotherapy is the first line of treatment. The 5-year survival rate for localized disease remains at 60–70% owing to advancement of neoadjuvant chemotherapy and surgery; however, there has been no significant improvement in the treatment of metastatic OS for the past 30 years with the survival rate remaining below 20%. A deeper understanding of the mechanisms underlying malignant progression of OS is required for discovering better therapeutic approaches to target metastatic OS. One of the hallmarks of malignant properties of cancer cells is their ability to survive in serumand anchorage-independent conditions and form spheres, since cancer cells must overcome proliferation arrest and cell death induced by serum deprivation as well as loss of anchorage (anoikis). In our recent study, we performed screening with a whole-genome human lentiviral shRNA library and identified an uncharacterized protein, transmembrane and immunoglobulin domain containing 3 (TMIGD3), as a factor whose downregulation significantly increased sphere-forming potential of OS cells. There are 2 isoforms of TMIGD3, i1 and i3, that share a common C-terminal region with an immunoglobulin (Ig)-like fold (Fig. 1). However, the functions of TMIGD3 i1 and i3 remain largely unknown. Moreover, TMIGD3 i1 shares its Nterminal region (first 117 amino acids in exon 1) with adenosine A3 receptor (A3AR, Fig. 1). A3AR, a well characterized Gi protein-associated G protein-coupled receptor (GPCR), is a member of the family of adenosine receptors including A1AR and A2AR, and is implicated in suppressing inflammation and cancer. Activation of A3AR by adenosine or its agonists results in inhibition of adenylyl cyclase and reduction in cyclic AMP (cAMP) production, leading to suppression of multiple signaling pathways, including the Wnt-b-catenin, MAPK (mitogen-activated protein kinase), and NF-kB (Fig. 1). To understand the roles of TMIGD3 on OS progression, we query for the functional similarities between A3AR and TMIGD3, although neither TMIGD3 i1 nor i3 has a typical GPCR structure. Downregulation of TMIGD3 or A3AR by multiple shRNAs significantly increases malignant properties of different OS cell lines, including proliferation, migration, and sphere formation, as well as tumor progression and metastasis in nude mice. Overexpression of A3AR and TMIGD3 i1, but not TMIGD3 i3, inhibits these malignant properties. Interestingly, similar to A3AR, TMIGD3 i1 suppresses activities of protein kinase A (PKA), PKB (also known as Akt), and NF-kB with minimal impacts on b-catenin and Erk1/2 activities, indicating that TMIGD3 i1 regulates overlapping signaling pathways with A3AR (Fig. 1). However, TMIGD3 i1 overexpression only partially rescues increase in sphere formation and cAMP production caused by A3AR knockdown. Moreover, unbiased luciferasebased pathway analyses reveal the presence of signaling pathways regulated by TMIGD3 distinct from A3AR. Thus, TMIGD3 i1 suppresses malignant properties of OS via both overlapping and non-overlapping pathways with A3AR. Protein expression of TMIGD3 in OS tissues is significantly lower than that in normal bone and lungs, similar to A3AR. Since transcription of both A3AR and TMIGD3 i1 is driven by the same promoter due to the common exon 1, expression of these proteins may be co-regulated. Consistently, high metastatic OS cell lines (KHOS/NP and MG63) tend to express lower levels of A3AR and TMIGD3 with lower IkB levels (indicating higher NF-kB activity), as compared with those in lowmetastatic cell lines (U2OS and Saos-2). It should be noted that shRNAs for TMIGD3 and an antibody for TMIGD3 used in our study could not discriminate between the 2 isoforms of i1 and i3. Hence, it would be crucial for identifying and using specific shRNAs and antibodies that can distinguish these isoforms in the future. The existence of 2 potential tumor suppressors having similar functions in the same chromosome locus (1p13.2) in humans is quite intriguing. According to the Ensembl genome browser (http://uswest.ensembl.org/index.html), orthologous genes for TMIGD3 i1 are present in the majority of primates, as well as in horse, armadillo, pika, and lesser hedgehog tenrecs, while exceptionally gibbon possesses only gene orthologous for TMIGD3 i3 but not TMIGD3 i1. However, these species do not possess gene orthologous for A3AR. Many other vertebrates have orthologous genes for A3AR, but not TMIGD3 i1. Thus, Human is the only species which has both genes for A3AR and TMIGD3 i1. It would be interesting to understand the reasoning behind the natural selection of TMIGD3 i1 vs A3AR during evolution and why humans have evolved to possess both genes.

MTBP inhibits the Erk1/2-Elk-1 signaling in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the prognosis of HCC patients, especially those with metastasis, remains extremely poor. This is partly due to unclear molecular mechanisms underlying HCC metastasis. Our previous study indicates that MDM2 Binding Protein (MTBP) suppresses migration and metastasis of HCC cells. However, signaling pathways regulated by MTBP remain unknown. To identify metastasis-associated signaling pathways governed by MTBP, we have performed unbiased luciferase reporter-based signal array analyses and found that MTBP suppresses the activity of the ETS-domain transcription factor Elk-1, a downstream target of Erk1/2 MAP kinases. MTBP also inhibits phosphorylation of Elk-1 and decreases mRNA expression of Elk-1 target genes. Reduced Elk-1 activity is caused by inhibited nuclear translocation of phosphorylated Erk1/2 (p-Erk) by MTBP and subsequent inhibition of Elk-1 phosphorylation. We also reveal that MTBP inhibits the interaction of p-Erk with importin-7/RanBP7 (IPO7), an importin family member which shuttles p-Erk into the nucleus, by binding to IPO7. Moreover, high levels of MTBP in human HCC tissues are correlated with cytoplasmic localization of p-Erk1/2. Our study suggests that MTBP suppresses metastasis, at least partially, by down-modulating the Erk1/2-Elk-1 signaling pathway, thus identifying a novel regulatory mechanism of HCC metastasis by regulating the subcellular localization of p-Erk.

Abstract B24: Regulation of mutant p53 stability by the mevalonate pathway-Hsp40-CHIP axis

Missense mutations in the p53 gene result in accumulation of dysfunctional p53 proteins in tumors with oncogenic gain-of-function activities, such as metastasis and chemotherapy resistance. Increasing evidence indicates that stabilization of mutant p53 (mutp53) in tumors is crucial for its oncogenic activities, while its knockdown reduces malignant progression. Thus, malignant properties of cancer cells are dependent on the presence of mutp53, providing a rationale to identify compounds that deplete mutp53 with minimal effects on wild-type p53 (wtp53). Toward this goal, we performed luciferase assay-based high-throughput screening of ~9,000 chemical compounds using cells expressing a chimeric fusion protein of p53R175H and a luciferase reporter. This screening led us to identify statins, a class of cholesterol-lowering drugs, as degradation inducers of conformational p53 mutants. Interestingly, statins showed minimal effects on wtp53 and DNA contact mutants. Moreover, CHIP ubiquitin ligase, but not MDM2, mediated statin-induced nuclear export, ubiquitination, and degradation of mutp53. Surprisingly, degradation of mutp53 by statins was independent of protein prenylation/lipidation or cholesterol synthesis, and specific reduction of mevalonte-5-phosphate (MVP) triggered mutp53 degradation. We also demonstrated that downregulation of a molecular chaperone Hsp40/DNAJA1 mirrored statin9s effects on mutp53, while its overexpression nullified them. Moreover, statin-induced reduction of MVP inhibited the DNAJA1-mutp53 interaction. Biologically, statins preferentially suppressed malignant properties of mutp53-expressing cancer cells in vitro and in vivo. Our study proposes a novel role of DNAJA1 in inhibiting CHIP-mediated degradation of mutp53 induced by statins and highlights the significance of p53 status in impacting the efficacy of statins in cancer therapy and of DNAJA1 as a viable target for inducing mutp53 degradation. Citation Format: Alejandro Parrales, Atul Ranjan, Swathi V. Iyer, Scott Weir, Anuradha Roy, Tomoo Iwakuma. Regulation of mutant p53 stability by the mevalonate pathway-Hsp40-CHIP axis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B24.

Abstract 3690: Metabolic regulation of mutant p53 stability by the mevalonate pathway

Introduction: Mutations in the p53 gene are mostly missense mutations and result in accumulation of dysfunctional p53 protein in tumors with oncogenic gain-of-function activities. Increasing evidence indicates that stabilization of mutant p53 (mutp53) in tumors is crucial for its oncogenic activities including tumor progression and drug resistance, while downregulation of mutp53 reduces oncogenicity of cancer cells. These observations suggest that malignant properties of cancer cells are dependent on the presence of mutp53, thus providing a rationale to identify compounds that deplete mutant p53 with little impact on wild-type p53. Experimental procedures: Toward this goal, we performed high throughput screens of chemical libraries (∼9,000 compounds) with Saos2 (p53 null) cells expressing a fusion protein of p53R175H and luciferase, using luciferase as a reporter. Summary of data: This screening led us to identify “statins”, a class of cholesterol-lowering medications, as compounds that induced degradation of p53R175H. We found that other inhibitors of the mevalonate pathway, such as 6-fluoromevalonate and zoledronic acid, failed to induce p53R175H degradation, while statin-mediated inhibition of HMG-CoA reductase and subsequent reduction in mevalonte-5-phosphate triggered p53R175H degradation. These results suggest that statin9s effect on p53R175H is specific and independent of protein prenylation/lipidation or cholesterol synthesis. Moreover, nuclear export of p53R175H was required for the statin-mediated degradation, which was mediated through an E3 ubiquitin ligase CHIP, but not MDM2. Interestingly, statins induced degradation of mainly conformational p53 mutants with minimal effects on the levels of wild-type p53 and DNA contact mutants. Conclusions: This is the first study demonstrating that mutp53 stability is regulated through a specific process of the mevalonate pathway, thereby providing a novel regulatory mechanism of mutp53 degradation. Our findings suggest that p53 mutation status in tumors may have an impact on efficacy of statins in cancer therapy. Citation Format: Tomoo Iwakuma, Atul Ranjan, Swathi V. Iyer, Subhash Padhye, Scott Weir, Anuradha Roy. Metabolic regulation of mutant p53 stability by the mevalonate pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3690.