Naproxen chemoprevention promotes immune activation in Lynch syndrome colorectal mucosa.

OBJECTIVE: Patients with Lynch syndrome (LS) are at markedly increased risk for colorectal cancer. It is being increasingly recognised that the immune system plays an essential role in LS tumour development, thus making an ideal target for cancer prevention. Our objective was to evaluate the safety, assess the activity and discover novel molecular pathways involved in the activity of naproxen as primary and secondary chemoprevention in patients with LS. DESIGN: We conducted a Phase Ib, placebo-controlled, randomised clinical trial of two dose levels of naproxen sodium (440 and 220 mg) administered daily for 6 months to 80 participants with LS, and a co-clinical trial using a genetically engineered mouse model of LS and patient-derived organoids (PDOs). RESULTS: Overall, the total number of adverse events was not different across treatment arms with excellent tolerance of the intervention. The level of prostaglandin E2 in the colorectal mucosa was significantly decreased after treatment with naproxen when compared with placebo. Naproxen activated different resident immune cell types without any increase in lymphoid cellularity, and changed the expression patterns of the intestinal crypt towards epithelial differentiation and stem cell regulation. Naproxen demonstrated robust chemopreventive activity in a mouse co-clinical trial and gene expression profiles induced by naproxen in humans showed perfect discrimination of mice specimens with LS and PDOs treated with naproxen and control. CONCLUSIONS: Naproxen is a promising strategy for immune interception in LS. We have discovered naproxen-induced gene expression profiles for their potential use as predictive biomarkers of drug activity. TRIAL REGISTRATION NUMBER: gov Identifier: NCT02052908.

Patched1 haploinsufficiency severely impacts intermediary metabolism in the skin of Ptch1+/-/ODC transgenic mice.

The study of dominantly heritable cancers has provided insights about tumor development. Gorlin syndrome (GS) is an autosomal dominant disorder wherein affected individuals develop multiple basal cell carcinomas (BCCs) of the skin. We developed a murine model of Ptch1 haploinsufficiency on an ornithine decarboxylase (ODC) transgenic background (Ptch1+/-/ODCt/C57BL/6) that is more sensitive to BCCs growth as compared with Ptch1+/+/ODCt/C57BL/6 littermates. Ptch1+/-/ODCt/C57BL/6 mice show an altered metabolic landscape in the phenotypically normal skin, including restricted glucose availability, restricted ribose/deoxyribose flow and NADPH production, an accumulation of α-ketoglutarate, aconitate, and citrate that is associated with reversal of the tricarboxylic acid cycle, coupled with increased ketogenic/lipogenic activity via acetyl-CoA, 3-hydroybutyrate, and cholesterol metabolites. Also apparent was an increased content/acetylation of amino-acids, glutamine and glutamate, in particular. Accordingly, metabolic alterations due to a single copy loss of Ptch1 in Ptch1+/-/ODCt/C57BL/6 heterozygous mice may provide insights about the cancer prone phenotype of BCCs in GS patients, including biomarkers/targets for early intervention.

ETHE1 overexpression promotes SIRT1 and PGC1α mediated aerobic glycolysis, oxidative phosphorylation, mitochondrial biogenesis and colorectal cancer.

Ethylmalonic Encephalopathy Protein 1 (ETHE1) is a sulfur dioxygenase that regulates cellular H2S levels. We previously demonstrated a significant increase of ETHE1 expression in "single-hit" colon epithelial cells from crypts of patients with Familial Adenomatous Polyposis (FAP). Here, we report elevated levels of ETHE1 expression and increased mitochondrial density occurring in-situ in phenotypically normal FAP colorectal mucosa. We also found that constitutive expression of ETHE1 increased aerobic glycolysis ("Warburg effect"), oxidative phosphorylation, and mitochondrial biogenesis in colorectal cancer (CRC) cell lines, thereby depleting H2S which relieved the inhibition of phosphodiesterase (PDE), and increased adenosine monophosphate (AMP) levels. This led to activation of the energy sensing AMP-activated protein kinase (AMPKp), Sirtuin1 (SIRT1) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a master regulator of mitochondrial biogenesis. By contrast, shRNA silencing of ETHE1 reduced PDE activity, AMPKp/SIRT1/PGC1α levels and mitochondrial biogenesis. Constitutive expression of ETHE1 accelerated both CRC cell xenograft and orthotopic patient derived xenograft CRC cell growth in vivo. Overall, our data nominate elevated ETHE1 expression levels as a novel biomarker and potential therapeutic target for the prevention of CRC tumorigenesis.

Global gene expression of histologically normal primary skin cells from BCNS subjects reveals "single-hit" effects that are influenced by rapamycin.

Studies of dominantly heritable cancers enabled insights about tumor progression. BCNS is a dominantly inherited disorder that is characterized by developmental abnormalities and postnatal neoplasms, principally BCCs. We performed an exploratory gene expression profiling of primary cell cultures derived from clinically unaffected skin biopsies of BCNS gene-carriers (PTCH1 +/-) and normal individuals. PCA and HC of untreated keratinocytes or fibroblasts failed to clearly distinguish BCNS samples from controls. These results are presumably due to the common suppression of canonical HH signaling in vitro. We then used a relaxed threshold (p-value <0.05, no FDR cut-off; FC 1.3) that identified a total of 585 and 857 genes differentially expressed in BCNS keratinocytes and fibroblasts samples, respectively. A GSEA identified pancreatic ß cell hallmark and mTOR signaling genes in BCNS keratinocytes, whereas analyses of BCNS fibroblasts identified gene signatures regulating pluripotency of stem cells, including WNT pathway. Significantly, rapamycin treatment (FDR<0.05), affected a total of 1411 and 4959 genes in BCNS keratinocytes and BCNS fibroblasts, respectively. In contrast, rapamycin treatment affected a total of 3214 and 4797 genes in normal keratinocytes and normal fibroblasts, respectively. The differential response of BCNS cells to rapamycin involved 599 and 1463 unique probe sets in keratinocytes and fibroblasts, respectively. An IPA of these genes in the presence of rapamycin pointed to hepatic fibrosis/stellate cell activation, and HIPPO signaling in BCNS keratinocytes, whereas mitochondrial dysfunction and AGRN expression were uniquely enriched in BCNS fibroblasts. The gene expression changes seen here are likely involved in the etiology of BCCs and they may represent biomarkers/targets for early intervention.

Erratum: Drug-Targeted Inhibition of Peroxisome Proliferator-Activated Receptor-gamma Enhances the Chemopreventive Effect of Anti-Estrogen Therapy.

[This corrects the article DOI: 10.18632/oncotarget.457.].

PPARs as determinants of the estrogen receptor lineage: use of synthetic lethality for the treatment of estrogen receptor-negative breast cancer.

THE DILEMMA: Estrogen receptora-negative (ER-) breast cancer lacks a specific critical target to control tumor progression. THE OBJECTIVE: To identify mechanisms that enable increased expression of the ER+ lineage in an otherwise ER- breast cancer. PREFACE: The nuclear receptor superfamily members PPARγ and PPARδ regulate gene expression associated with a multitude of pathways, including intermediary metabolism, angiogenesis, proliferation and inflammation (see reviews [1-3]). Recent developments using transgenic and knockout mice, as well as pharmacologic intervention with PPARγ and PPARδ agonists, have revealed a previously unknown relationship between PPARγ suppression and PPARδ activation that leads to the appearance of ER+ tumors, enabling a synthetic lethality approach by anti-ER therapy. The ability to selectively affect the ER+ lineage by modulating PPARγ and PPARδ activity represents a new clinical paradigm and opportunity to treat ER- cancer with PPARγ and PPARδ modulating agents, ultimately rendering them more responsive to adjuvant therapy.

Naproxen Inhibits UVB-induced Basal Cell and Squamous Cell Carcinoma Development in Ptch1+/- /SKH-1 Hairless Mice.

Naproxen possesses anti-proliferative and pro-apoptotic effects besides its known anti-inflammatory functions. Here, we demonstrate the anticancer effects of naproxen against UVB-induced basal cell carcinoma (BCCs) and squamous cell carcinoma (SCCs) in a highly susceptible murine model of UVB carcinogenesis. Naproxen significantly inhibited UVB-induced BCCs and SCCs in this model. Tumor number and volume were significantly decreased (P < 0.005 and P < 0.05, respectively). Inhibition in UVB-induced SCCs and BCCs was 77% and 86%, respectively, which was associated with reduced PCNA and cyclin D1 and increased apoptosis. As expected, inflammation-related iNOS, COX-2 and nuclear NFκBp65 were also diminished by naproxen treatment. Residual tumors excised from naproxen-treated animal were less invasive and showed reduced expression of epithelial-mesenchymal transition (EMT) markers N-cadherin, Vimentin, Snail and Twist with increased expression of E-cadherin. In BCC and SCC cells, naproxen-induced apoptosis and activated unfolded protein response (UPR) signaling with increased expression of ATF4, p-eIF2α and CHOP. Employing iRNA-based approaches, we found that naproxen-induced apoptosis was regulated by CHOP as sensitivity of these cutaneous neoplastic cells for apoptosis was significantly diminished by ablating CHOP. In summary, these data show that naproxen is a potent inhibitor of UVB-induced skin carcinogenesis. ER stress pathway protein CHOP may play an important role in inducing apoptosis in cancer cells.

Haploinsufficiency in tumor predisposition syndromes: altered genomic transcription in morphologically normal cells heterozygous for VHL or TSC mutation.

Tumor suppressor genes and their effector pathways have been identified for many dominantly heritable cancers, enabling efforts to intervene early in the course of disease. Our approach on the subject of early intervention was to investigate gene expression patterns of morphologically normal "one-hit" cells before they become hemizygous or homozygous for the inherited mutant gene which is usually required for tumor formation. Here, we studied histologically non-transformed renal epithelial cells from patients with inherited disorders that predispose to renal tumors, including von Hippel-Lindau (VHL) disease and Tuberous Sclerosis (TSC). As controls, we studied histologically normal cells from non-cancerous renal epithelium of patients with sporadic clear cell renal cell carcinoma (ccRCC). Gene expression analyses of VHLmut/wt or TSC1/2mut/wt versus wild-type (WT) cells revealed transcriptomic alterations previously implicated in the transition to precancerous renal lesions. For example, the gene expression changes in VHLmut/wt cells were consistent with activation of the hypoxia response, associated, in part, with the "Warburg effect". Knockdown of any remaining VHL mRNA using shRNA induced secondary expression changes, such as activation of NFκB and interferon pathways, that are fundamentally important in the development of RCC. We posit that this is a general pattern of hereditary cancer predisposition, wherein haploinsufficiency for VHL or TSC1/2, or potentially other tumor susceptibility genes, is sufficient to promote development of early lesions, while cancer results from inactivation of the remaining normal allele. The gene expression changes identified here are related to the metabolic basis of renal cancer and may constitute suitable targets for early intervention.

Simultaneous targeting of 5-LOX-COX and ODC block NNK-induced lung adenoma progression to adenocarcinoma in A/J mice.

Lung cancer is the leading cause of cancer deaths worldwide. Targeting complementary pathways will achieve better treatment efficacy than a single agent high-dose strategy that could increase risk of side effects and tumor resistance. To target COX-2, 5-LOX, and ODC simultaneously, we tested the effects of a dual 5-LOX-COX inhibitor, licofelone, and an ODC inhibitor, DFMO, alone and in combination, on NNK-induced lung tumors in female A/J mice. Seven-week-old mice were treated with NNK (10 µmol/mouse, single dose, i.p.) and randomized to different treatment groups. Three weeks after injection, mice were fed control or experimental diets (DFMO 1500/3000 ppm, licofelone 200/400 ppm, or a low-dose combination of 1500 ppm DFMO and 200 ppm licofelone) for 17 or 34 weeks. Both agents significantly inhibited tumor formation in a dose-dependent manner. As anticipated more adenomas and adenocarcinomas were observed at 17 and 34 weeks, respectively. Importantly, low dose combination of DFMO and licofelone showed more pronounced effects at 17 or 34 weeks in inhibiting the total tumor formation (~60%, p < 0.0001) and adenocarcinoma (~65%, p < 0.0001) compared to individual high dose of DFMO (~44% and 46%, p < 0.0001) and licofelone (~48% and 55%, p < 0.0001). DFMO and combination-treated mice lung tumors exhibited modulated ODC pathway components (Oat, Oaz, SRM, SMS, and SAT, p < 0.05) along with decreased proliferation (PCNA, Cyclin D1 and Cyclin A) and increased expression of p53, p21 and p27 compared to mice fed control diet. Both DFMO and licofelone significantly inhibited tumor inflammatory markers. Our findings suggest that a low-dose combined treatment targeting inflammation and polyamine synthesis may provide effective chemoprevention.

Targeting cancer stem cells with p53 modulators.

Cancer stem cells (CSC) typically over-express aldehyde dehydrogenase (ALDH). Thus, ALDHbright tumor cells represent targets for developing novel cancer prevention/treatment interventions. Loss of p53 function is a common genetic event during cancer development wherein small molecular weight compounds (SMWC) that restore p53 function and reverse tumor growth have been identified. Here, we focused on two widely studied p53 SMWC, CP-31398 and PRIMA-1, to target ALDHbright CSC in human breast, endometrial and pancreas carcinoma cell lines expressing mutant or wild type (WT) p53. CP-31398 and PRIMA-1 significantly reduced CSC content and sphere formation by these cell lines in vitro. In addition, these agents were more effective in vitro against CSC compared to cisplatin and gemcitabine, two often-used chemotherapeutic agents. We also tested a combinatorial treatment in methylcholantrene (MCA)-treated mice consisting of p53 SMWC and p53-based vaccines. Yet using survival end-point analysis, no increased efficacy in the presence of either p53 SMWC alone or with vaccine compared to vaccine alone was observed. These results may be due, in part, to the presence of immune cells, such as activated lymphocytes expressing WT p53 at levels comparable to some tumor cells, wherein further increase of p53 expression by p53 SMWC may alter survival of these immune cells and negatively impact an effective immune response. Continuous exposure of mice to MCA may have also interfered with the action of these p53 SMWC, including potential direct interaction with MCA. Nonetheless, the effect of p53 SMWC on CSC and cancer treatment remains of great interest.

Targeting mTOR and p53 Signaling Inhibits Muscle Invasive Bladder Cancer In Vivo.

Urothelial tumors, accompanied by mutations of the tumor suppressor protein TP53 and dysregulation of mTOR signaling, are frequently associated with aggressive growth and invasiveness. We investigated whether targeting these two pathways would inhibit urothelial tumor growth and progression. Six-week-old transgenic UPII-SV40T male mice (n = 15/group) were fed control diet (AIN-76A) or experimental diets containing mTOR inhibitor (rapamycin, 8 or 16 ppm), p53 stabilizing agent [CP31398 (CP), 150 ppm], or a combination. Mice were euthanized at 40 weeks of age. Urinary bladders were collected and evaluated to determine tumor weight and histopathology. Each agent alone, and in combination, significantly inhibited tumor growth. Treatment with rapamycin alone decreased tumor weight up to 67% (P < 0.0001). Similarly, CP showed approximately 77% (P < 0.0001) suppression of tumor weight. The combination of low-dose rapamycin and CP led to approximately 83% (P < 0.0001) inhibition of tumor weight. There was no significant difference in tumor weights between rapamycin and CP treatments (P > 0.05). However, there was a significant difference between 8 ppm rapamycin and the combination treatment. Tumor invasion was also significantly inhibited in 53% (P < 0.005) and 66% (P < 0.0005) mice after 8 ppm and 16 ppm rapamycin, respectively. However, tumor invasion was suppressed in 73% (P < 0.0001) mice when CP was combined with 8 ppm rapamycin. These results suggest that targeting two or more pathways achieve better treatment efficacy than a single-agent high-dose strategy that could increase the risk of side effects. A combination of CP and rapamycin may be a promising method of inhibiting muscle-invasive urothelial transitional cell carcinoma.

TP53 modulating agent, CP-31398 enhances antitumor effects of ODC inhibitor in mouse model of urinary bladder transitional cell carcinoma.

Mutations of the tumor suppressor p53 and elevated levels of polyamines are known to play key roles in urothelial tumorigenesis. We investigated the inhibition of polyamines biosynthesis and the restoration of p53 signaling as a possible means of preventing muscle invasive urothelial tumors using DFMO, an ODC-inhibiting agent, and CP-31398 (CP), a p53 stabilizing agent. Transgenic UPII-SV40T male mice at 6weeks age (n=15/group) were fed control diet (AIN-76A) or experimental diets containing DFMO (1000 and 2000 ppm) or 150 ppm CP or both. At 40 weeks of age, all mice were euthanized and urinary bladders were evaluated to determine tumor weight and histopathology. Low-dose DFMO had a moderate significant inhibitory effect on tumor growth (38%, P<0.02) and tumor invasion (23%). High-dose DFMO had a 47% tumor inhibition (P<0.0001) and 40% inhibition tumor invasion. There was no significant difference between 1000 and 2000 ppm doses of DFMO (P>0.05). CP at 150 ppm alone had a strong inhibitory effect on tumor growth by 80% (P<0.0001); however, no effect on tumor invasion was observed. Interestingly, the combination of DFMO (1000 ppm) and CP (150 ppm) led to significant decrease in tumor weight (70%, P<0.0001) and tumor invasion (62.5%; P<0.005). Molecular analysis of the urothelial tumors suggested a modulation of polyamine biosynthesis, proliferation, cell cycle regulators resulting from the use of these agents. These results suggest that targeting two or more pathways could be an effective approach for chemoprevention. A combination of CP and DFMO appears to be a promising strategy for urothelial TCC prevention.