A second-generation eIF4A RNA helicase inhibitor exploits translational reprogramming as a vulnerability in triple-negative breast cancer.

In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications.

Cleavage of the V-ATPase associated prorenin receptor is mediated by PACE4 and is essential for growth of prostate cancer cells.

Phosphatase and tensin homolog (PTEN) mutation is common in prostate cancer during progression to metastatic and castration resistant forms. We previously reported that loss of PTEN function in prostate cancer leads to increased expression and secretion of the Prorenin Receptor (PRR) and its soluble processed form, the soluble Prorenin Receptor (sPRR). PRR is an essential factor required for proper assembly and activity of the vacuolar-ATPase (V-ATPase). The V-ATPase is a rotary proton pump required for the acidification of intracellular vesicles including endosomes and lysosomes. Acidic vesicles are involved in a wide range of cancer related pathways such as receptor mediated endocytosis, autophagy, and cell signalling. Full-length PRR is cleaved at a conserved consensus motif (R-X-X-R↓) by a member of the proprotein convertase family to generate sPRR, and a smaller C-terminal fragment, designated M8.9. It is unclear which convertase processes PRR in prostate cancer cells and how processing affects V-ATPase activity. In the current study we show that PRR is predominantly cleaved by PACE4, a proprotein convertase that has been previously implicated in prostate cancer. We further demonstrate that PTEN controls PRR processing in mouse tissue and controls PACE4 expression in prostate cancer cells. Furthermore, we demonstrate that PACE4 cleavage of PRR is needed for efficient V-ATPase activity and prostate cancer cell growth. Overall, our data highlight the importance of PACE4-mediated PRR processing in normal physiology and prostate cancer tumorigenesis.

A Renaissance for Oncolytic Adenoviruses?

In the 1990s, adenovirus became one of the first virus types to be genetically engineered to selectively destroy cancer cells. In the intervening years, the field of "oncolytic viruses" has slowly progressed and culminated in 2015 with the FDA approval of Talimogene laherparepvec, a genetically engineered herpesvirus, for the treatment of metastatic melanoma. Despite the slower progress in translating oncolytic adenovirus to the clinic, interest in the virus remains strong. Among all the clinical trials currently using viral oncolytic agents, the largest proportion of these are using recombinant adenovirus. Many trials are currently underway to use oncolytic virus in combination with immune checkpoint inhibitors (ICIs), and early results using oncolytic adenovirus in this manner are starting to show promise. Many of the existing strategies to engineer adenoviruses were designed to enhance selective tumor cell replication without much regard to interactions with the immune system. Adenovirus possesses a wide range of viral factors to attenuate both innate anti-viral pathways and immune cell killing. In this review, we summarize the strategies of oncolytic adenoviruses currently in clinical trials, and speculate how the mutational backgrounds of these viruses may impact upon the efficacy of these agents in oncolytic and immunotherapy. Despite decades of research on human adenoviruses, the interactions that these viruses have with the immune system remains one of the most understudied aspects of the virus and needs to be improved to rationally design the next generation of engineered viruses.

Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer.

CDC20 is a coactivator of the anaphase promoting complex/cyclosome (APC/C) and is essential for mitotic progression. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC), which prevents premature separation of sister chromatids and aneuploidy in daughter cells. Although overexpression of CDC20 is common in many cancers, oncogenic mutations have never been identified in humans. Using whole-exome sequencing, we identified heterozygous missense CDC20 variants (L151R and N331K) that segregate with ovarian germ cell tumors in two families. Functional characterization showed these mutants retain APC/C activation activity but have impaired binding to BUBR1, a component of the SAC. Expression of L151R and N331K variants promoted mitotic slippage in HeLa cells and primary skin fibroblasts derived from carriers. Generation of mice carrying the N331K variant using CRISPR-Cas9 showed that, although homozygous N331K mice were nonviable, heterozygotes displayed accelerated oncogenicity of Myc-driven cancers. These findings highlight an unappreciated role for CDC20 variants as tumor-promoting genes. SIGNIFICANCE: Two germline CDC20 missense variants that segregate with cancer in two families compromise the spindle assembly checkpoint and lead to aberrant mitotic progression, which could predispose cells to transformation. See related commentary by Villarroya-Beltri and Malumbres, p. 3432.

The mTORC1/S6K/PDCD4/eIF4A Axis Determines Outcome of Mitotic Arrest.

mTOR is a serine/threonine kinase and a master regulator of cell growth and proliferation. Raptor, a scaffolding protein that recruits substrates to mTOR complex 1 (mTORC1), is known to be phosphorylated during mitosis, but the significance of this phosphorylation remains largely unknown. Here we show that raptor expression and mTORC1 activity are dramatically reduced in cells arrested in mitosis. Expression of a non-phosphorylatable raptor mutant reactivates mTORC1 and significantly reduces cytotoxicity of the mitotic poison Taxol. This effect is mediated via degradation of PDCD4, a tumor suppressor protein that inhibits eIF4A activity and is negatively regulated by the mTORC1/S6K pathway. Moreover, pharmacological inhibition of eIF4A is able to enhance the effects of Taxol and restore sensitivity in Taxol-resistant cancer cells. These findings indicate that the mTORC1/S6K/PDCD4/eIF4A axis has a pivotal role in the death versus slippage decision during mitotic arrest and may be exploited clinically to treat tumors resistant to anti-mitotic agents.

Elevated V-ATPase Activity Following PTEN Loss Is Required for Enhanced Oncogenic Signaling in Breast Cancer.

PTEN loss-of-function contributes to hyperactivation of the PI3K pathway and to drug resistance in breast cancer. Unchecked PI3K pathway signaling increases activation of the mechanistic target of rapamycin complex 1 (mTORC1), which promotes tumorigenicity. Several studies have suggested that vacuolar (H+)-ATPase (V-ATPase) complex activity is regulated by PI3K signaling. In this study, we showed that loss of PTEN elevated V-ATPase activity. Enhanced V-ATPase activity was mediated by increased expression of the ATPase H+ transporting accessory protein 2 (ATP6AP2), also known as the prorenin receptor (PRR). PRR is cleaved into a secreted extracellular fragment (sPRR) and an intracellular fragment (M8.9) that remains associated with the V-ATPase complex. Reduced PTEN expression increased V-ATPase complex activity in a PRR-dependent manner. Breast cancer cell lines with reduced PTEN expression demonstrated increased PRR expression. Similarly, PRR expression became elevated upon PTEN deletion in a mouse model of breast cancer. Interestingly, concentration of sPRR was elevated in the plasma of patients with breast cancer and correlated with tumor burden in HER2-enriched cancers. Moreover, PRR was essential for proper HER2 receptor expression, localization, and signaling. PRR knockdown attenuated HER2 signaling and resulted in reduced Akt and ERK 1/2 phosphorylation, and in lower mTORC1 activity. Overall, our study demonstrates a mechanism by which PTEN loss in breast cancer can potentiate multiple signaling pathways through upregulation of the V-ATPase complex. IMPLICATIONS: Our study contributed to the understanding of the role of the V-ATPase complex in breast cancer cell tumorigenesis and provided a potential biomarker in breast cancer.

The Role of Apoptin in Chicken Anemia Virus Replication.

Apoptin is the Vp3 protein of chicken anemia virus (CAV), which infects the thymocytes and erythroblasts in young chickens, causing chicken infectious anemia and immunosuppression. Apoptin is highly studied for its ability to selectively induce apoptosis in human tumor cells and, thus, is a protein of interest in anti-tumor therapy. CAV apoptin is known to localize to different subcellular compartments in transformed and non-transformed cells, depending on the DNA damage response, and the phosphorylation of several identified threonine residues. In addition, apoptin interacts with molecular machinery such as the anaphase promoting complex/cyclosome (APC/C) to inhibit the cell cycle and induce arrest in G2/M phase. While these functions of apoptin contribute to the tumor-selective effect of the protein, they also provide an important fundamental framework to apoptin's role in viral infection, pathogenesis, and propagation. Here, we reviewed how the regulation, localization, and functions of apoptin contribute to the viral life cycle and postulated its importance in efficient replication of CAV. A model of the molecular biology of infection is critical to informing our understanding of CAV and other related animal viruses that threaten the agricultural industry.

V-ATPase-associated prorenin receptor is upregulated in prostate cancer after PTEN loss.

Phosphatase and tensin homolog (PTEN) tumor suppressor protein loss is common in prostate cancer (PCa). PTEN loss increases PI3K/Akt signaling, which promotes cell growth and survival. To find secreted biomarkers of PTEN loss, a proteomic screen was used to compare secretomes of cells with and without PTEN expression. We showed that PTEN downregulates Prorenin Receptor (PRR) expression and secretion of soluble Prorenin Receptor (sPRR) in PCa cells and in mouse. PRR is an accessory protein required for assembly of the vacuolar ATPase (V-ATPase) complex. V-ATPase is required for lysosomal acidification, amino acid sensing, efficient mechanistic target of Rapamycin complex 1 (mTORC1) activation, and ß-Catenin signaling. On PCa tissue microarrays, PRR expression displayed a positive correlation with Akt phosphorylation. Moreover, PRR expression was required for proliferation of PCa cells by maintaining V-ATPase function. Further, we provided evidence for a potential clinical role for PRR expression and sPRR concentration in differentiating low from high Gleason grade PCa. Overall, the current study unveils a mechanism by which PTEN can inhibit tumor growth. Lower levels of PRR result in attenuated V-ATPase activity and reduced PCa cell proliferation.

Mutations in ANAPC1, Encoding a Scaffold Subunit of the Anaphase-Promoting Complex, Cause Rothmund-Thomson Syndrome Type 1.

Rothmund-Thomson syndrome (RTS) is an autosomal-recessive disorder characterized by poikiloderma, sparse hair, short stature, and skeletal anomalies. Type 2 RTS, which is defined by the presence of bi-allelic mutations in RECQL4, is characterized by increased cancer susceptibility and skeletal anomalies, whereas the genetic basis of RTS type 1, which is associated with juvenile cataracts, is unknown. We studied ten individuals, from seven families, who had RTS type 1 and identified a deep intronic splicing mutation of the ANAPC1 gene, a component of the anaphase-promoting complex/cyclosome (APC/C), in all affected individuals, either in the homozygous state or in trans with another mutation. Fibroblast studies showed that the intronic mutation causes the activation of a 95 bp pseudoexon, leading to mRNAs with premature termination codons and nonsense-mediated decay, decreased ANAPC1 protein levels, and prolongation of interphase. Interestingly, mice that were heterozygous for a knockout mutation have an increased incidence of cataracts. Our results demonstrate that deficiency in the APC/C is a cause of RTS type 1 and suggest a possible link between the APC/C and RECQL4 helicase because both proteins are involved in DNA repair and replication.

Reciprocal Regulation between 53BP1 and the Anaphase-Promoting Complex/Cyclosome Is Required for Genomic Stability during Mitotic Stress.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that targets substrates for degradation to promote mitotic progression. Here, we show that the DNA damage response protein 53BP1 contains conserved KEN boxes that are required for APC/C-dependent degradation in early mitosis. Mutation of the 53BP1 KEN boxes stabilized the protein and extended mitotic duration, whereas 53BP1 knockdown resulted in a shorter and delayed mitosis. Loss of 53BP1 increased APC/C activity, and we show that 53BP1 is a direct APC/C inhibitor. Although 53BP1 function is not absolutely required for normal cell cycle progression, knockdown was highly toxic in combination with mitotic spindle poisons. Moreover, chemical inhibition of the APC/C was able to rescue the lethality of 53BP1 loss. Our findings reveal a reciprocal regulation between 53BP1 and APC/C that is required for response to mitotic stress and may contribute to the tumor-suppressor functions of 53BP1.

Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication.

UNLABELLED: Chicken anemia virus (CAV) is a single-stranded circular DNA virus that carries 3 genes, the most studied of which is the gene encoding VP3, also known as apoptin. This protein has been demonstrated to specifically kill transformed cells while leaving normal cells unharmed in a manner that is independent of p53 status. Although the mechanistic basis for this differential activity is unclear, it is evident that the subcellular localization of the protein is important for the difference. In normal cells, apoptin exists in filamentous networks in the cytoplasm, whereas in transformed cells, apoptin is present in the nucleus and appears as distinct foci. We have previously demonstrated that DNA damage signaling through the ataxia telangiectasia mutated (ATM) pathway induces the translocation of apoptin from the cytoplasm to the nucleus, where it induces apoptosis. We found that apoptin contains four checkpoint kinase consensus sites and that mutation of either threonine 56 or 61 to alanine restricts apoptin to the cytoplasm. Furthermore, treatment of tumor cells expressing apoptin with inhibitors of checkpoint kinase 1 (Chk1) and Chk2 causes apoptin to localize to the cytoplasm. Importantly, silencing of Chk2 rescues cancer cells from the cytotoxic effects of apoptin. Finally, treatment of virus-producing cells with Chk inhibitor protects them from virus-mediated toxicity and reduces the titer of progeny virus. Taken together, our results indicate that apoptin is a sensor of DNA damage signaling through the ATM-Chk2 pathway, which induces it to migrate to the nucleus during viral replication. IMPORTANCE: The chicken anemia virus (CAV) protein apoptin is known to induce tumor cell-specific death when expressed. Therefore, understanding its regulation and mechanism of action could provide new insights into tumor cell biology. We have determined that checkpoint kinase 1 and 2 signaling is important for apoptin regulation and is a likely feature of both tumor cells and host cells producing virus progeny. Inhibition of checkpoint signaling prevents apoptin toxicity in tumor cells and attenuates CAV replication, suggesting it may be a future target for antiviral therapy.

Deletion of the putative tumor suppressor gene, G0s2, does not affect progression of Eµ-Myc driven lymphomas in mice.

Several recent reports have suggested that the G0/G1 switch gene 2 (G0S2) is a potential tumor suppressor in leukemia. Here we show that deletion of the G0s2 gene in mouse does not affect the latency of cancer progression in the Eµ-Myc model of lymphoma. Our findings do not rule out the possibility that G0S2 may be playing a role in other forms of leukemia, but clearly show that the commonly used Eµ-Myc transgenic is not the correct model to conduct studies on G0s2.

Inhibition of adipose triglyceride lipase (ATGL) by the putative tumor suppressor G0S2 or a small molecule inhibitor attenuates the growth of cancer cells.

The G0/G1 switch gene 2 (G0S2) is methylated and silenced in a wide range of human cancers. The protein encoded by G0S2 is an endogenous inhibitor of lipid catabolism that directly binds adipose triglyceride lipase (ATGL). ATGL is the rate-limiting step in triglyceride metabolism. Although the G0S2 gene is silenced in cancer, the impact of ATGL in the growth and survival of cancer cells has never been addressed. Here we show that ectopic expression of G0S2 in non-small cell lung carcinomas (NSCL) inhibits triglyceride catabolism and results in lower cell growth. Similarly, knockdown of ATGL increased triglyceride levels, attenuated cell growth and promoted apoptosis. Conversely, knockdown of endogenous G0S2 enhanced the growth and invasiveness of cancer cells. G0S2 is strongly induced in acute promyelocytic leukemia (APL) cells in response to all trans retinoic acid (ATRA) and we show that inhibition of ATGL in these cells by G0S2 is required for efficacy of ATRA treatment. Our data uncover a novel tumor suppressor mechanism by which G0S2 directly inhibits activity of a key intracellular lipase. Our results suggest that elevated ATGL activity may be a general property of many cancer types and potentially represents a novel target for chemotherapy.

Targeting B16 tumors in vivo with peptide-conjugated gold nanoparticles.

This study examines the effects of polyethylene glycol (PEG) and peptide conjugation on the biodistribution of ultrasmall (2.7 nm) gold nanoparticles in mice bearing B16 melanoma allografts. Nanoparticles were delivered intravenously, and biodistribution was measured at specific timepoints by organ digestion and inductively coupled plasma mass spectrometry. All major organs were examined. Two peptides were tested: the cyclic RGD peptide (cRGD, which targets integrins); and a recently described peptide derived from the myxoma virus. We found the greatest specific tumor delivery using the myxoma peptide, with or without PEGylation. Un-PEGylated cRGD performed poorly, but PEGylated RGD showed a significant transient collection in the tumor. Liver and kidney were the primary targets of all constructs. None of the particles were able to cross the blood-brain barrier. Although it was able to deliver Au to B16 cells, the myxoma peptide did not show any cytotoxic activity against these cells, in contrast to previous reports. These results indicate that the effect of passive targeting by PEGylation and active targeting by peptides can be independent or combined, and that they should be evaluated on a case-by-case basis when designing new nanosystems for targeted therapies. Both myxoma peptide and cRGD should be considered for specific targeting to melanoma, but a thorough investigation of the cytotoxicity of the myxoma peptide to different cell lines remains to be performed.

Intratumoral gold-doxorubicin is effective in treating melanoma in mice.

Intratumoral injection of ultra-small gold nanoparticles (AuNPs) conjugated to doxorubicin (Au-Dox) is effective against both murine B16 and human SK-MEL-28 tumors in mice. Au-Dox suppresses growth of B16 tumors in immunocompetent mice by >70% for at least 19 days. In SK-MEL-28 xenografts, Au-Dox suppresses tumor growth almost completely for >13 weeks, while tumors treated with Dox alone demonstrate accelerated growth after 10 weeks. Histological analysis shows significant apoptosis and necrosis in Au-Dox treated tumors. Intratumoral injection is significantly more effective than intravenous injection, which leads to significant accumulation in liver and kidney with sub-therapeutic concentrations of Au-Dox. However, IV injection does not lead to significant damage in non-target organs, so improved targeting should permit this mode of delivery with little risk of systemic toxicity. The current construct is suitable for tumors accessible to intratumoral injection and represents a viable approach doxorubicin-resistant solid tumors. FROM THE CLINICAL EDITOR: Drug resistance is a significant problem in the fight against cancer. The authors describe a new approach in combating drug resistance in tumor cells by conjugating ultrasmall gold nanoparticles to doxorubicin. They tested the efficacy in in-vivo models using two melanoma cell lines. The promising results obtained from intra-tumoral injections contribute a way in future drug designs showing that conjugation to nanoparticles could lead to more effective and synergistic killing of tumor cells.

Analysis by live imaging of effects of the adenovirus E4orf4 protein on passage through mitosis of H1299 tumor cells.

The adenovirus E4orf4 protein expressed at high levels kills cancer cells but not normal human primary cells. Previous studies suggested that disruption of processes that regulate mitosis may underlie E4orf4 toxicity. Here we have used live imaging to show that E4orf4 induces a slowed defective transit through mitosis, exhibiting a delay or often failure in cytokinesis that may account for an accumulation of G1 tetraploids in the population of dying E4orf4-expressing cells.

Interaction of adenovirus type 5 E4orf4 with the nuclear pore subunit Nup205 is required for proper viral gene expression.

UNLABELLED: Adenovirus type 5 E4orf4 is a multifunctional protein that regulates viral gene expression. The activities of E4orf4 are mainly mediated through binding to protein phosphatase 2A (PP2A). E4orf4 recruits target phosphoproteins into complexes with PP2A, resulting in dephosphorylation of host factors, such as SR splicing factors. In the current study, we utilized immunoprecipitation followed by mass spectrometry to identify novel E4orf4-interacting proteins. In this manner we identified Nup205, a component of the nuclear pore complex (NPC) as an E4orf4 interacting partner. The arginine-rich motif (ARM) of E4orf4 was required for interaction with Nup205 and for nuclear localization of E4orf4. ARMs are commonly found on viral nuclear proteins, and we observed that Nup205 interacts with three different nuclear viral proteins containing ARMs. E4orf4 formed a trimolecular complex containing both Nup205 and PP2A. Furthermore, Nup205 complexed with E4orf4 was hypophosphorylated, suggesting that the protein is specifically targeted for dephosphorylation. An adenovirus mutant that does not express E4orf4 (Orf4(-)) displayed elevated early and reduced late gene expression relative to that of the wild type. We observed that knockdown of Nup205 resulted in the same phenotype as that of the Orf4(-) virus, suggesting that the proteins function as a complex to regulate viral gene expression. Furthermore, knockdown of Nup205 resulted in a more than a 4-fold reduction in the replication of wild-type adenovirus. Our data show for first time that Ad5 E4orf4 interacts with and modifies the NPC and that Nup205-E4orf4 binding is required for normal regulation of viral gene expression and viral replication. IMPORTANCE: Nuclear pore complexes (NPCs) are highly regulated conduits in the nuclear membrane that control transport of macromolecules between the nucleus and cytoplasm. Viruses that replicate in the nucleus must negotiate the NPC during nuclear entry, and viral DNA, mRNA, and proteins must then be exported from the nucleus. Several types of viruses restructure the NPC to facilitate replication, and the current study shows that adenovirus type 5 (Ad5) utilizes a novel mechanism to modify NPC function. We demonstrate that a subunit of the NPC, Nup205, is a phosphoprotein that is actively dephosphorylated by the Ad5-encoded protein E4orf4. Moreover, Nup205 is required by Ad5 to regulate viral gene expression and efficient viral replication. Nup205 is a nonstructural subunit that is responsible for the gating functions of the NPC, and this study suggests for the first time that the NPC is regulated by phosphorylation both during normal physiology and viral infection.

Multifaceted regulation of somatic cell reprogramming by mRNA translational control.

Translational control plays a pivotal role in the regulation of the pluripotency network in embryonic stem cells, but its effect on reprogramming somatic cells to pluripotency has not been explored. Here, we show that eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs), which are translational repressors, have a multifaceted effect on the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs). Loss of 4E-BP expression attenuates the induction of iPSCs at least in part through increased translation of p21, a known inhibitor of somatic cell reprogramming. However, MEFs lacking both p53 and 4E-BPs show greatly enhanced reprogramming resulting from a combination of reduced p21 transcription and enhanced translation of endogenous mRNAs such as Sox2 and Myc and can be reprogrammed through the expression of only exogenous Oct4. Thus, 4E-BPs exert both positive and negative effects on reprogramming, highlighting the key role that translational control plays in regulating this process.

Adenovirus E4orf4 protein-induced death of p53-/- H1299 human cancer cells follows a G1 arrest of both tetraploid and diploid cells due to a failure to initiate DNA synthesis.

The adenovirus E4orf4 protein selectively kills human cancer cells independently of p53 and thus represents a potentially promising tool for the development of novel antitumor therapies. Previous studies suggested that E4orf4 induces an arrest or a delay in mitosis and that both this effect and subsequent cell death rely largely on an interaction with the B55 regulatory subunit of protein phosphatase 2A. In the present report, we show that the death of human H1299 lung carcinoma cells induced by expression of E4orf4 is typified not by an accumulation of cells arrested in mitosis but rather by the presence of both tetraploid and diploid cells that are arrested in G1 because they are unable to initiate DNA synthesis. We believe that these E4orf4-expressing cells eventually die by various processes, including those resulting from mitotic catastrophe.

p53 inhibits angiogenesis by inducing the production of Arresten.

Several types of collagen contain cryptic antiangiogenic noncollagenous domains that are released upon proteolysis of extracellular matrix (ECM). Among those is Arresten, a collagen-derived antiangiogenic factor (CDAF) that is processed from α1 collagen IV. However, the conditions under which Arresten is released from collagen IV in vivo or whether the protein functions in tumor suppressor pathways remain unknown. Here, we show that p53 induces the expression of α1 collagen IV and release of Arresten-containing fragments from the ECM. Comparison of the transcriptional activation of COL4A1 with other CDAF-containing genes revealed that COL4A1 is a major antiangiogenic gene induced by p53 in human adenocarinoma cells. p53 directly activated transcription of the COL4A1 gene by binding to an enhancer region 26 kbp downstream of its 3' end. p53 also stabilized the expression of full-length α1 collagen IV by upregulation of α(II) prolyl-hydroxylase and increased the release of Arresten in the ECM through a matrix metalloproteinase (MMP)-dependent mechanism. The resulting upregulation of α1 collagen IV and production of Arresten by the tumor cells significantly inhibited angiogenesis and limited tumor growth in vivo. Furthermore, we show that immunostaining of Arresten correlated with p53 status in human prostate cancer specimens. Our findings, therefore, link the production of Arresten to the p53 tumor suppressor pathway and show a novel mechanism through which p53 can inhibit angiogenesis.