Impaired ribosome biogenesis checkpoint activation induces p53-dependent MCL-1 degradation and MYC-driven lymphoma death.

MYC-driven B-cell lymphomas are addicted to increased levels of ribosome biogenesis (RiBi), offering the potential for therapeutic intervention. However, it is unclear whether inhibition of RiBi suppresses lymphomagenesis by decreasing translational capacity and/or by p53 activation mediated by the impaired RiBi checkpoint (IRBC). Here we generated Eµ-Myc lymphoma cells expressing inducible short hairpin RNAs to either ribosomal protein L7a (RPL7a) or RPL11, the latter an essential component of the IRBC. The loss of either protein reduced RiBi, protein synthesis, and cell proliferation to similar extents. However, only RPL7a depletion induced p53-mediated apoptosis through the selective proteasomal degradation of antiapoptotic MCL-1, indicating the critical role of the IRBC in this mechanism. Strikingly, low concentrations of the US Food and Drug Administration-approved anticancer RNA polymerase I inhibitor Actinomycin D (ActD) dramatically prolonged the survival of mice harboring Trp53+/+;Eµ-Myc but not Trp53-/-;Eµ-Myc lymphomas, which provides a rationale for treating MYC-driven B-cell lymphomas with ActD. Importantly, the molecular effects of ActD on Eµ-Myc cells were recapitulated in human B-cell lymphoma cell lines, highlighting the potential for ActD as a therapeutic avenue for p53 wild-type lymphoma.

Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy.

Macroautophagy/autophagy is suppressed by MTOR (mechanistic target of rapamycin kinase) and is an anticancer target under active investigation. Yet, MTOR-regulated autophagy remains incompletely mapped. We used proteomic profiling to identify proteins in the MTOR-autophagy axis. Wild-type (WT) mouse cell lines and cell lines lacking individual autophagy genes (Atg5 or Ulk1/Ulk2) were treated with an MTOR inhibitor to induce autophagy and cultured in media with either glucose or galactose. Mass spectrometry proteome profiling revealed an elevation of known autophagy proteins and candidates for new autophagy components, including CALCOCO1 (calcium binding and coiled-coil domain protein 1). We show that CALCOCO1 physically interacts with MAP1LC3C, a key protein in the machinery of autophagy. Genetic deletion of CALCOCO1 disrupted autophagy of the endoplasmic reticulum (reticulophagy). Together, these results reveal a role for CALCOCO1 in MTOR-regulated selective autophagy. More generally, the resource generated by this work provides a foundation for establishing links between the MTOR-autophagy axis and proteins not previously linked to this pathway. Abbreviations: ATG: autophagy-related; CALCOCO1: calcium binding and coiled-coil domain protein 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain protein 2; CLIR: MAP1LC3C-interacting region; CQ: chloroquine; KO: knockout; LIR: MAP1LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLN: MLN0128 ATP-competitive MTOR kinase inhibitor; MTOR: mechanistic target of rapamycin kinase; reticulophagy: selective autophagy of the endoplasmic reticulum; TAX1BP1/CALCOCO3: TAX1 binding protein 1; ULK: unc 51-like autophagy activating kinase; WT: wild-type.

Hepatic Ago2-mediated RNA silencing controls energy metabolism linked to AMPK activation and obesity-associated pathophysiology.

RNA silencing inhibits mRNA translation. While mRNA translation accounts for the majority of cellular energy expenditure, it is unclear if RNA silencing regulates energy homeostasis. Here, we report that hepatic Argonaute 2 (Ago2)-mediated RNA silencing regulates both intrinsic energy production and consumption and disturbs energy metabolism in the pathogenesis of obesity. Ago2 regulates expression of specific miRNAs including miR-802, miR-103/107, and miR-148a/152, causing metabolic disruption, while simultaneously suppressing the expression of genes regulating glucose and lipid metabolism, including Hnf1ß, Cav1, and Ampka1. Liver-specific Ago2-deletion enhances mitochondrial oxidation and ATP consumption associated with mRNA translation, which results in AMPK activation, and improves obesity-associated pathophysiology. Notably, hepatic Ago2-deficiency improves glucose metabolism in conditions of insulin receptor antagonist treatment, high-fat diet challenge, and hepatic AMPKα1-deletion. The regulation of energy metabolism by Ago2 provides a novel paradigm in which RNA silencing plays an integral role in determining basal metabolic activity in obesity-associated sequelae.

Mitochondrial Complex I Activity Is Required for Maximal Autophagy.

Cells adapt to nutrient and energy deprivation by inducing autophagy, which is regulated by the mammalian target of rapamycin (mTOR) and AMP-activated protein kinases (AMPKs). We found that cell metabolism significantly influences the ability to induce autophagy, with mitochondrial complex I function being an important factor in the initiation, amplitude, and duration of the response. We show that phenformin or genetic defects in complex I suppressed autophagy induced by mTOR inhibitors, whereas autophagy was enhanced by strategies that increased mitochondrial metabolism. We report that mTOR inhibitors significantly increased select phospholipids and mitochondrial-associated membranes (MAMs) in a complex I-dependent manner. We attribute the complex I autophagy defect to the inability to increase MAMs, limiting phosphatidylserine decarboxylase (PISD) activity and mitochondrial phosphatidylethanolamine (mtPE), which support autophagy. Our data reveal the dynamic and metabolic regulation of autophagy.

Phenformin-Induced Mitochondrial Dysfunction Sensitizes Hepatocellular Carcinoma for Dual Inhibition of mTOR.

Purpose: Hepatocellular carcinoma (HCC) ranks second in cancer mortality and has limited therapeutic options. We recently described the synergistic effect of allosteric and ATP-site competitive inhibitors against the mTOR for the treatment of HCC. However, such inhibitors induce hyperglycemia and increase mitochondrial efficiency. Here we determined whether the mitochondrial complex I inhibitor phenformin could reverse both side effects, impose an energetic stress on cancer cells, and suppress the growth of HCC.Experimental Design: Human HCC cell lines were used in vitro to access the signaling and energetic impact of mTOR inhibitors and phenformin, either alone or in combination. Next, the therapeutic utility of these drugs alone or in combination was investigated preclinically in human orthotopic tumors implanted in mice, by analyzing their impact on the tumor burden and overall survival.Results: We found phenformin caused mitochondrial dysfunction and fragmentation, inducing a compensatory shift to glycolysis. In contrast, dual inhibition of mTOR impaired cell growth and glycolysis, while increasing mitochondrial fusion and efficiency. In a mouse model of human HCC, dual inhibition of mTOR, together with phenformin, was highly efficacious in controlling tumor burden. However, more strikingly, pretreatment with phenformin sensitized tumors to dual inhibition of mTOR, leading to a dramatic improvement in survival.Conclusions: Treatment of HCC cells in vitro with the biguanide phenformin causes a metabolic shift to glycolysis, mitochondrial dysfunction and fragmentation, and dramatically sensitizes orthotopic liver tumors to dual inhibition of mTOR. We therefore propose this therapeutic approach should be tested clinically in HCC. Clin Cancer Res; 24(15); 3767-80. ©2018 AACR.

mTOR Kinase Inhibition Effectively Decreases Progression of a Subset of Neuroendocrine Tumors that Progress on Rapalog Therapy and Delays Cardiac Impairment.

Inhibition of mTOR signaling using the rapalog everolimus is an FDA-approved targeted therapy for patients with lung and gastroenteropancreatic neuroendocrine tumors (NET). However, patients eventually progress on treatment, highlighting the need for additional therapies. We focused on pancreatic NETs (pNET) and reasoned that treatment of these tumors upon progression on rapalog therapy, with an mTOR kinase inhibitor (mTORKi), such as CC-223, could overcome a number of resistance mechanisms in tumors and delay cardiac carcinoid disease. We performed preclinical studies using human pNET cells in vitro and injected them subcutaneously or orthotopically to determine tumor progression and cardiac function in mice treated with either rapamycin alone or switched to CC-223 upon progression. Detailed signaling and RNA sequencing analyses were performed on tumors that were sensitive or progressed on mTOR treatment. Approximately 57% of mice bearing pNET tumors that progressed on rapalog therapy showed a significant decrease in tumor volume upon a switch to CC-223. Moreover, mice treated with an mTORKi exhibited decreased cardiac dilation and thickening of heart valves than those treated with placebo or rapamycin alone. In conclusion, in the majority of pNETs that progress on rapalogs, it is possible to reduce disease progression using an mTORKi, such as CC-223. Moreover, CC-223 had an additional transient cardiac benefit on valvular fibrosis compared with placebo- or rapalog-treated mice. These results provide the preclinical rationale to further develop mTORKi clinically upon progression on rapalog therapy and to further test their long-term cardioprotective benefit in those NET patients prone to carcinoid syndrome. Mol Cancer Ther; 16(11); 2432-41. ©2017 AACR.

A Phase Ib Study of the Dual PI3K/mTOR Inhibitor Dactolisib (BEZ235) Combined with Everolimus in Patients with Advanced Solid Malignancies.

BACKGROUND: The combination of everolimus and the imidazoquinoline derivative, BEZ235 (dactolisib), a dual PI3K/mTOR inhibitor, demonstrated synergy in a preclinical model. OBJECTIVE: To establish clinical feasibility, a phase Ib dose-escalation trial investigating safety and pharmacokinetics of this combination in patients with advanced tumors was performed. PATIENTS AND METHODS: BEZ235 was orally administered daily in escalating doses of 200, 400, and 800 mg along with everolimus at 2.5 mg daily in 28-day cycles. Nineteen patients were enrolled. Adverse events and tumor responses were evaluated using CTCAE v4.0 and RECIST 1.1, respectively. Pharmacokinetic analyses were performed. RESULTS: Common toxicities observed included fatigue, diarrhea, nausea, mucositis, and elevated liver enzymes. No confirmed responses were observed. BEZ235 pharmacokinetics exhibited dose-proportional increases in Cmax and AUC0-24 over the three doses, with high inter-individual variability. Non-compartmental and population pharmacokinetic-based simulations indicated significant increases in everolimus Cmax and AUC0-24 on day 28 and decreased clearance to 13.41 L/hr. CONCLUSIONS: The combination of BEZ235 and everolimus demonstrated limited efficacy and tolerance. BEZ235 systemic exposure increased in a dose-proportional manner while oral bioavailability was quite low, which may be related to gastrointestinal-specific toxicity. The changes in steady-state pharmacokinetics of everolimus with BEZ235 highlight potential drug-drug interactions when these two drugs are administered together. Clinicaltrials.gov: NCT01508104.

A Phase I Study of Cixutumumab (IMC-A12) in Combination with Temsirolimus (CCI-779) in Children with Recurrent Solid Tumors: A Children's Oncology Group Phase I Consortium Report.

PURPOSE: To determine the MTD, dose-limiting toxicities (DLT), pharmacokinetics, and biologic effects of cixutumumab administered in combination with temsirolimus to children with refractory solid tumors. EXPERIMENTAL DESIGN: Cixutumumab and temsirolimus were administered intravenously once every 7 days in 28-day cycles. Pharmacokinetic and biology studies, including assessment of mTOR downstream targets in peripheral blood mononuclear cells, were performed during the first cycle. RESULTS: Thirty-nine patients, median age 11.8 years (range, 1-21.5), with recurrent solid or central nervous system tumors were enrolled, of whom 33 were fully assessable for toxicity. There were four dose levels, which included two dose reductions and a subsequent intermediated dose escalation: (i) IMC-A12 6 mg/kg, temsirolimus 15 mg/m(2); (ii) IMC-A12 6 mg/kg, temsirolimus 10 mg/m(2); (iii) IMC-A12 4 mg/kg, temsirolimus 8 mg/m(2); and (iv) IMC-A12 6 mg/kg, temsirolimus 8 mg/m(2). Mucositis was the predominant DLT. Other DLTs included hypercholesterolemia, fatigue, thrombocytopenia, and increased alanine aminotransferase. Target inhibition (decreased S6K1 and PAkt) in peripheral blood mononuclear cells was noted at all dose levels. Marked interpatient variability in temsirolimus pharmacokinetic parameters was noted. At 8 mg/m(2), the median temsirolimus AUC was 2,946 ng • h/mL (range, 937-5,536) with a median sirolimus AUC of 767 ng • h/mL (range, 245-3,675). CONCLUSIONS: The recommended pediatric phase II doses for the combination of cixutumumab and temsirolimus are 6 mg/kg and 8 mg/m(2), respectively.

Loss of tumor suppressor RPL5/RPL11 does not induce cell cycle arrest but impedes proliferation due to reduced ribosome content and translation capacity.

Humans have evolved elaborate mechanisms to activate p53 in response to insults that lead to cancer, including the binding and inhibition of Hdm2 by the 60S ribosomal proteins (RPs) RPL5 and RPL11. This same mechanism appears to be activated upon impaired ribosome biogenesis, a risk factor for cancer initiation. As loss of RPL5/RPL11 abrogates ribosome biogenesis and protein synthesis to the same extent as loss of other essential 60S RPs, we reasoned the loss of RPL5 and RPL11 would induce a p53-independent cell cycle checkpoint. Unexpectedly, we found that their depletion in primary human lung fibroblasts failed to induce cell cycle arrest but strongly suppressed cell cycle progression. We show that the effects on cell cycle progression stemmed from reduced ribosome content and translational capacity, which suppressed the accumulation of cyclins at the translational level. Thus, unlike other tumor suppressors, RPL5/RPL11 play an essential role in normal cell proliferation, a function cells have evolved to rely on in lieu of a cell cycle checkpoint.

5S ribosomal RNA is an essential component of a nascent ribosomal precursor complex that regulates the Hdm2-p53 checkpoint.

Recently, we demonstrated that RPL5 and RPL11 act in a mutually dependent manner to inhibit Hdm2 and stabilize p53 following impaired ribosome biogenesis. Given that RPL5 and RPL11 form a preribosomal complex with noncoding 5S ribosomal RNA (rRNA) and the three have been implicated in the p53 response, we reasoned they may be part of an Hdm2-inhibitory complex. Here, we show that small interfering RNAs directed against 5S rRNA have no effect on total or nascent levels of the noncoding rRNA, though they prevent the reported Hdm4 inhibition of p53. To achieve efficient inhibition of 5S rRNA synthesis, we targeted TFIIIA, a specific RNA polymerase III cofactor, which, like depletion of either RPL5 or RPL11, did not induce p53. Instead, 5S rRNA acts in a dependent manner with RPL5 and RPL11 to inhibit Hdm2 and stabilize p53. Moreover, depletion of any one of the three components abolished the binding of the other two to Hdm2, explaining their common dependence. Finally, we demonstrate that the RPL5/RPL11/5S rRNA preribosomal complex is redirected from assembly into nascent 60S ribosomes to Hdm2 inhibition as a consequence of impaired ribosome biogenesis. Thus, the activation of the Hdm2-inhibitory complex is not a passive but a regulated event, whose potential role in tumor suppression has been recently noted.

Growth control and ribosomopathies.

Ribosome biogenesis and protein synthesis are two of the most energy consuming processes in a growing cell. Moreover, defects in their molecular components can alter the pattern of gene expression. Thus it is understandable that cells have developed a surveillance system to monitor the status of the translational machinery. Recent discoveries of causative mutations and deletions in genes linked to ribosome biogenesis have defined a group of similar pathologies termed ribosomopathies. Over the past decade, much has been learned regarding the relationship between growth control and ribosome biogenesis. The discovery of extra-ribosomal functions of several ribosome proteins and their regulation of p53 levels has provided a link from ribosome impairment to cell cycle regulation. Yet, evidence suggesting p53 and/or Hdm2 independent pathways also exists. In this review, we summarize recent advances in understanding the mechanisms underlying the pathologies of ribosomopathies and discuss the relationship between ribosome production and tumorigenesis.

mTOR inhibitors synergize on regression, reversal of gene expression, and autophagy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) affects more than half a million people worldwide and is the third most common cause of cancer deaths. Because mammalian target of rapamycin (mTOR) signaling is up-regulated in 50% of HCCs, we compared the effects of the U.S. Food and Drug Administration-approved mTOR-allosteric inhibitor, RAD001, with a new-generation phosphatidylinositol 3-kinase/mTOR adenosine triphosphate-site competitive inhibitor, BEZ235. Unexpectedly, the two drugs acted synergistically in inhibiting the proliferation of cultured HCC cells. The synergistic effect closely paralleled eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) dephosphorylation, which is implicated in the suppression of tumor cell proliferation. In a mouse model approximating human HCC, the drugs in combination, but not singly, induced a marked regression in tumor burden. However, in the tumor, BEZ235 alone was as effective as the combination in inhibiting 4E-BP1 phosphorylation, which suggests that additional target(s) may also be involved. Microarray analyses revealed a large number of genes that reverted to normal liver tissue expression in mice treated with both drugs, but not either drug alone. These analyses also revealed the down-regulation of autophagy genes in tumors compared to normal liver. Moreover, in HCC patients, altered expression of autophagy genes was associated with poor prognosis. Consistent with these findings, the drug combination had a profound effect on UNC51-like kinase 1 (ULK1) dephosphorylation and autophagy in culture, independent of 4E-BP1, and in parallel induced tumor mitophagy, a tumor suppressor process in liver. These observations have led to an investigator-initiated phase 1B-2 dose escalation trial with RAD001 combined with BEZ235 in patients with HCC and other advanced solid tumors.

Interleukin-6 receptor enhances early colonization of the murine omentum by upregulation of a mannose family receptor, LY75, in ovarian tumor cells.

One of the earliest metastatic events in human ovarian cancer, tumor spread to the omentum, may be influenced by expression of interleukin 6 (IL6) and its cognate receptor (IL6Rα). Previous reports have shown that IL6 and IL6Rα expression is elevated in the serum and ascites of patients with ovarian cancer and that this can influence in vitro processes such as cell survival, proliferation and migration. In this study, overexpression of IL6Rα, and to a lesser extent IL6, enhanced tumor growth on the omentum. Moreover, adherence to plastic and to peritoneal extracellular matrix components was enhanced in tumor cells overexpressing IL6 or IL6Rα. Host production of IL6 and IL6Rα was also sufficient to influence tumor adherence to the omentum. Expression of LY75/CD205/DEC205, a collagen-binding mannose family receptor, was directly influenced by IL6Rα expression. Blocking LY75 with antibody reduced the adherence of tumor cells overexpressing IL6Rα to matrices in vitro and to the omentum. The association between IL6Rα expression and LY75 expression has not been previously reported, and the promotion of cellular adherence is a novel role for LY75. These studies indicate that overexpression of LY75 may be an additional mechanism by which IL6 signaling influences the progression of ovarian cancer, and suggests that blocking LY75 could be a valuable clinical strategy for reducing the early metastasis of ovarian cancer.

Differences in wound healing in mice with deficiency of IL-6 versus IL-6 receptor.

IL-6 modulates immune responses and is essential for timely wound healing. As the functions mediated by IL-6 require binding to its specific receptor, IL-6Ralpha, it was expected that mice lacking IL-6Ralpha would have the same phenotype as IL-6-deficient mice. However, although IL-6Ralpha-deficient mice share many of the inflammatory deficits seen in IL-6-deficient mice, they do not display the delay in wound healing. Surprisingly, mice with a combined deficit of IL-6 and IL-6Ralpha, or IL-6-deficient mice treated with an IL-6Ralpha-blocking Ab, showed improved wound healing relative to mice with IL-6 deficiency, indicating that the absence of the receptor contributed to the restoration of timely wound healing, rather than promiscuity of IL-6 with an alternate receptor. Wounds in mice lacking IL-6 showed delays in macrophage infiltration, fibrin clearance, and wound contraction that were not seen in mice lacking IL-6Ralpha alone and were greatly reduced in mice with a combined deficit of IL-6 and IL-6Ralpha. MAPK activation-loop phosphorylation was elevated in wounds of IL-6Ralpha-deficient mice, and treatment of wounds in these mice with the MEK inhibitor U0126 resulted in a delay in wound healing suggesting that aberrant ERK activation may contribute to improved healing. These findings underscore a deeper complexity for IL-6Ralpha function in inflammation than has been recognized previously.

A novel, human Atg13 binding protein, Atg101, interacts with ULK1 and is essential for macroautophagy.

Macroautophagy is an intracellular, vesicle-mediated mechanism for the sequestration and ultimate lysosomal degradation of cytoplasmic proteins, organelles and macromolecules. The macroautophagy process and many of the autophagy-specific (Atg) proteins are remarkably well conserved in higher eukaryotes. In yeast, the Atg1 kinase complex includes Atg1, Atg13, Atg17, and at least four other interacting proteins, some of which are phosphorylated in a TOR-dependent manner, placing the Atg1 signaling complex downstream of a major nutrient-sensing pathway. Atg1 orthologs, including mammalian unc-51-like kinase 1 (ULK1), have been identified in higher eukaryotes and have been functionally linked to autophagy. This suggests that other components of the Atg1 complex exist in higher eukaryotes. Recently, a putative human Atg13 ortholog, FLJ20698, was identified by gapped-BLAST analysis. We show here that FLJ20698 (Atg13) is a ULK1-interacting phosphoprotein that is essential for macroautophagy. Furthermore, we identify a novel, human Atg13-interacting protein, FLJ11773, which we have termed Atg101. Atg101 is essential for autophagy and interacts with ULK1 in an Atg13-dependent manner. Additionally, we present evidence that intracellular localization of the ULK1 complex is regulated by nutrient conditions. Finally, we demonstrate that Atg101 stabilizes the expression of Atg13 in the cell, suggesting that Atg101 contributes to Atg13 function by protecting Atg13 from proteasomal degradation. Therefore, the identification of the novel protein, Atg101, and the validation of Atg13 and Atg101 as ULK1-interacting proteins, suggests an Atg1 complex is involved in the induction of macroautophagy in mammalian cells.

The GST-BHMT assay and related assays for autophagy.

The endpoint of the autophagic process is the breakdown of delivered cytoplasmic cargo in lysosomes. Therefore, assays based on degradation of cargo are of particular interest in that they can measure regulation of the entire autophagic process, including changes in cargo delivery and breakdown in the lytic compartment. Betaine homocysteine methyltransferase (BHMT) is one of many cytosolic proteins found in the mammalian autophagosome, and delivery of BHMT to the lysosome results in its proteolysis to discrete fragments under certain conditions. Making use of these observations, the GST-BHMT assay was developed as an endpoint, cargo-based autophagy assay. Using this assay as a starting point, additional cargo-based assays have been developed with the potential to measure autophagic degradation of specific subcellular compartments. Here we describe the development and validation of these assays.

Macroautophagy-dependent, intralysosomal cleavage of a betaine homocysteine methyltransferase fusion protein requires stable multimerization.

Cargo-based assays have proven invaluable in the study of macroautophagy in yeast and mammalian cells. Proteomic analysis of autolysosomes identified the metabolic enzyme, betaine homocysteine methyltransferase (BHMT), as a potential cargo-based, end-point marker for mammalian macroautophagy. To test whether degradation of BHMT can be used to measure macroautophagic flux in mammalian cells, we created a BHMT fusion protein (GST-BHMT) that demonstrates starvation-induced, site-specific fragmentation in a variety of cell lines. Subcellular fractionation studies show that the GST-BHMT fragment co-fractionates with vesicles containing lysosomal and autolysosomal markers. Furthermore, both pharmacological inhibitors of macroautophagy and depletion of macroautophagy-specific proteins reduce accumulation of the fragment. In the course of these studies, we observed that fragmentation of GST-BHMT did not occur in forms of the reporter with truncation or point mutations that destabilize oligomerization. Since stable oligomerization of BHMT is essential for its catalytic activity, a point mutation known to ablate BHMT activity was tested. We show that accumulation of the GST-BHMT fragment is not impaired in a catalytically inactive mutant, indicating that selective proteolysis of GST-BHMT requires stable quaternary structure independent of effects on activity. Also, the loss of fragmentation observed in the oligomerization deficient mutants does not seem to be due to a defect of sequestration and lysosomal loading, suggesting that disruption of stable quaternary structure affects the ability of a lysosomal protease to cleave the newly-delivered cargo. Finally, we propose that the cargo-based GST-BHMT assay will be a valuable addition to existing macroautophagy assays in mammalian cells.

Macrophages mediate inflammation-enhanced metastasis of ovarian tumors in mice.

The tumor microenvironment is known to have a profound effect on tumor progression in a highly context-specific manner. We have investigated whether peritoneal inflammation plays a causative role in ovarian tumor metastasis, a poorly understood process. Implantation of human ovarian tumor cells into the ovaries of severe combined immunodeficient mice resulted in peritoneal inflammation that corresponds temporally with tumor cell dissemination from the ovaries. Enhancement of the inflammatory response with thioglycolate accelerated the development of ascites and metastases. Suppression of inflammation with acetyl salicylic acid delayed ascites development and reduced tumor implant formation. A similar prometastatic effect for inflammation was observed when tumor cells were injected directly into the peritoneum of severe combined immunodeficient mice, and in a syngeneic immunocompetent mouse model. Inflammation-modulating treatments did not affect primary tumor development or in vitro tumor cell growth. Depletion of peritoneal macrophages, but not neutrophils or natural killer cells, reduced tumor progression, as assessed by ascites formation and peritoneal metastasis. We conclude that inflammation facilitates ovarian tumor metastasis by a mechanism largely mediated by macrophages, and which may involve stromal vascular endothelial growth factor production. The confirmation of these findings in immunocompetent mice suggests relevance to human disease. Identifying the mechanisms by which macrophages contribute to tumor metastasis may facilitate the development of new therapies specifically targeting immune cell products in the tumor microenvironment.

p63 overexpression induces the expression of Sonic Hedgehog.

p63 and p73 are members of the p53 protein family and have been shown to play an important role in cell death, development, and tumorigenesis. In particular, p63 has been shown to be involved in the maintenance of epidermal stem cells and in the stratification of the epidermis. Sonic Hedgehog (Shh) is a morphogen that has also been implicated to play a role in epithelial stem cell proliferation and in the development of organs. Recently, Shh has also been shown to play an important role in the progression of a variety of cancers. In this report, we show that p63 and p73 but not p53 overexpression induces Shh expression. In particular, p63gamma and p63beta (both TA and DeltaN isoforms) and TAp73beta isoform induce Shh. Expression of Shh was found to be significantly reduced in mouse embryo fibroblasts obtained from p63-/- mice. The naturally occurring p63 mutant TAp63gamma(R279H) and the tumor suppressor protein p14(ARF) inhibited the TAp63gamma-mediated transactivation of Shh. The region -228 to -102 bp of Shh promoter was found to be responsive to TAp63gamma-induced transactivation and TAp63gamma binds to regions within the Shh promoter in vivo. The results presented in this study implicate p63 in the regulation of the Shh signaling pathway.