Zn deficiency-induced inflammation and oral-esophageal carcinogenesis

Transcription

1 Zn deficiency-induced inflammation and oral-esophageal carcinogenesis Louise Y.Y. Fong, Ph.D. Department of Pharmacology & Experimental Therapeutics Kimmel Cancer Center Thomas Jefferson University

2 Inflammation as the 7th hallmark of cancer Hanahan and Weinberg Cell 2000; Mantovani Nature

3 Oral-esophageal squamous cell cancers Major cause of cancer deaths worldwide Oral Cancer, tongue being the major site Incidence is increasing; high mortality Esophageal cancer (ESCC and EAC) 5 year-survival is ~10% Risk factors: alcohol consumption, tobacco, HPV Epidemiologic and clinical studies have long implicated Zn-deficiency in the pathogenesis of these cancers. 3

4 Zn has many roles in biological processes An essential nutrient Required for the activity of > 200 enzymes Required for proper immune function Required for the conformation of many transcription factors that control cell proliferation, differentiation, and signaling pathways Zn is capable of undergoing rapid ligand exchange Thus, Zn-deficiency predisposes to disease by adversely affecting immune system, by increasing oxidative stress, and by increasing the generation of inflammatory cytokines 4

5 Zn Sources in Foods Who are Zn deficient? Good sources: red meat and seafood Poor sources: whole grain and most vegetables, because of the presence of phytate that binds Zn and inhibits its absorption A person subsisting on a cereal and vegetarian diet is likely to have a low Zn intake The elderly are vulnerable to ZD owing to inadequate diets and/or intestinal malabsorption, and therefore, susceptible to age-related diseases such as infections and cancer Conditioned ZD can occur in individuals with diseases associated with impaired intestinal absorption, chronic inflammatory bowel diseases, rheumatoid arthritis, diabetes, alcoholism, stress, and cancer RDA: ~12-15 mg In the US: Zn intake is <50% of RDA for ~10% of the population In the developing world, dietary ZD may affect >2 billion people 5

6 ZD rat/mouse cancer models Reproduce human Zn deficiency In rodents, a Zn-deficient diet creates a precancerous condition in the upper digestive tract by inducing cell proliferation and gene expression changes NMBA is used to induce esophageal tumors NQO is used to induce tongue tumors Both carcinogens induced tumors that are morphologically similar to human ESCC and LSCC, progressing histopathologically from hyperplasia to mild/severe dysplasia, then to carcinoma in situ, and finally to invasive squamous cell carcinoma 6

7 Esophageal cell proliferation: BrDU labeling Zn-sufficient (pair-fed) Zn-sufficient (ad libitum fed) Zn-deficient (ad libitum fed) 7 Fong et al. Carcinogenesis, 1996

8 Effects of Zn deficiency (ZD) and Zn replenishment (ZR) lingual cell proliferation : PCNA immunohistochemistry 8 Fong et al. JNCI, 2005

9 NQO induces tumors at multiple sites (tongue, esophagus, and forestomach) in ZD rats 9

10 NQO induces tumors at multiple sites in the upper aerodigestive tract in ZD rat 10

11 Zn-Replenishment reduces cancer reversing cell proliferation increasing apoptosis correcting abnormal gene expression 11

12 Zn replenishment initiated hours after NMBA treatment: reduction of esophageal carcinogenesis Animal group* (time between NMBA treatment and initiation of zinc replenishment) Tumor incidence (%) No. of tumors per esophagus (95% confidence interval) ZD (not replenished) 14/15 (93) 3.1 (1.9 to 4.3) ZR1 (1 h) 3/37 (8) 0.08 (0 to 0.17) ZR24 (24 h) 5/37 (14) 0.14 (0.03 to 0.25) ZR72 (72 h) 5/26 (19) 0.27 (0 to 0.54) ZR432 (432 h) 19/40 (48) 0.65 (0.40 to 0.90) *ZD = zinc-deficient; zinc-replenished (ZR1, ZR24, ZR72, and ZR432 rats were replenished, respectively, at 1, 24, 72, and 432 hours after NMBA dosing). Fong et al. JNCI

13 ZD Hyperkeratosis, hyperplastic, FHLs, sporadic single apoptotic cells Morphologic changes in esophageal epithelia from Znreplenished (ZR) rats ZR1 Clusters of apoptotic cells, suprabasal cells show condensed chromatin 13

14 Apoptosis and cell proliferation in ZD and ZR esophagi at 24, 30, and 48 hr after treatment with NMBA 14

15 What are the mechanisms underlying the pro-tumorigenic effects of ZD? We hypothesized that ZD promotes esophageal carcinogenesis by inducing the activity of cyclooxygenase-2 (COX-2). 15

16 COX-2 An inducible enzyme that catalyzes the formation of prostaglandins from arachidonic acid. Overexpressed in a variety of premalignant and malignant cancers, including esophageal and oral cancer Quickly induced by factors that are implicated in carcinogenesis, for example, growth factors, inflammatory stimuli, oncogenes, and tumor promoters. Overexpression of COX-2 enhances cell proliferation, inhibits apoptosis, modulates angiogenesis, thereby contributing to carcinogenesis. COX-2 deletion in Apc knockout mice greatly reduces intestinal polyp formation, providing genetic evidence that COX-2 plays a role in tumorigenesis 16

17 COX-2 mrna expression in ZD and ZR esophagus 17

18 COX-2 protein expression in tongue and esophagus from Znmodulated rats 18

19 Spatial localization of COX-2 protein in Zn-deficient rat esophagus 19

20 Gene expression profiling of hyperplastic ZD rat esophagus to identify key biologic differences affected by nutritional Zn deficiency in the absence of carcinogenic insult Study design 5 wk 2 d Age ~3-4 wk Zn Zn Deficient Zn Sufficient Zn Replenished All Animals Sacrificed And Microarray Analysis Performed Taccioli et al., Gastroenterology

21 Expression profiling of preneoplastic ZD esophagus: Affymetrix GeneChip rat genome array 21

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23 Verification of array data by real-time qrt-pcr and immunoblot assays 23

24 Expression Data Analysis EASE (Expression Analysis Systematic Explorer) software was used to identify biological pathways overrepresented among the 103 genes. Response to external stimulus pathway (7 genes: S100a8, Cdkn1a, Gja1, Mx2, Ppp2r1a, Sectm1, and Serpinb3) was the only significantly overrepresented pathway among the upregulated genes (EASE score = 0.02). This result supports S100a8 as a relevant marker in ZDinduced esophageal hyperplasia. 24

25 S100A8/A9 Encode the S100 family member of Ca binding proteins that also bind Zn Originally discovered as immunogenic protein expressed and secreted by neutrophils Important mediators in inflammation Overexpressed in a variety of human cancers, including skin SCC, ESCC, Barrett s esophagus, lung, prostate cancer Play a key role in inflammation-associated cancers 25

26 Model of a novel activation network in inflammation and cancer 26 Gebhardt C et al. Biochem Pharmacol 72: , 2006

27 Zn modulates the link between S100A8-RAGE interaction and downstream nuclear factor NF- COX-2 signaling 27

28 S100A8 expression in human and mouse ESCC 28

29 S100A8/A9 mrna expression in esophageal carcinogenesis

30 Conclusions 1. Dietary Zn deficiency induces a proinflammatory gene signature in rat esophagus with strong upregulation of S100a8 and S100a9, associated with hyperplasia. 2. Zn replenishment reverses overexpression of the proinflammatory mediator S100a8 and esophageal preneoplasia. 3. Zn regulates S100A8 expression and modulates the link between S100A8-RAGE interaction and downstream NF- /COX-2 signaling. 4. These data provide the first evidence that Zn regulates an inflammatory pathway in early esophageal carcinogenesis and its reversal. 30

31 Gene expression changes during Zn deficiency driven esophageal tumor development Study design 15 wk 5 wk 5 wk 1 wk 6 wk 3 wk Zn Deficient Age ~3-4 wk Zn Zn Sufficient Zn Replenished NMBA NMBA Some Animals Sacrificed And Microarray Analysis Performed NMBA Remaining Animals Sacrificed And Tumor Incidence / Microarray Analyses Performed Tumor incidence at 15 wk Unpublished data 31

32 Summary 1. Transcriptome profiling of Zn-deficient rat esophagi during NMBAinduced esophageal tumor development reveals a cancer-related inflammatory gene signature associated with neoplasia. 2. In addition to S100a8 and S100a9 overexpression, several cancerassociated inflammation genes, namely, CXC chemokines Cxcl5, Cxcl1, Cxcl2, the inflammatory enzyme Ptgs2 (Cox-2) and interleukin Il1b and ll17f are strongly upregulated in ZD esophagi. 3. ZR attenuates the expression of these inflammatory genes and suppresses tumorigenesis. 4. The data demonstrate that prolonged Zn-deficiency causes chronic inflammation in the esophagus that fuels carcinogenesis. ZR modulates inflammatory responses and inhibits tumor growth. 40

33 Targeted cancer therapy Increasingly cancers are treated with drugs that target specific pathways shown to be of pathogenetic significance. Targeting the COX-2 pathway COX-2 selective inhibitors are actively being tested in clinical trials for the prevention of several cancers including, colorectal, esophageal adenocarcinoma, and head and neck cancer. 41

34 Summary of previous study: In ZD rats pharmacololgic COX-2 inhibition by the drug celecoxib did not prevent tongue carcinogenesis Instead of being protected, Zn-deficient COX-2 null mice developed significantly greater tumor multiplicity and forestomach carcinoma incidence than wild-type controls. Fong et al., Int J Cancer

35 Effect of dietary Zn-deficiency on NMBA-induced forestomach carcinogenesis in COX-2 deficient mice 43

36 LTA4H overexpression in ZD:COX-2 -/- forestomach in the absence of carcinogen treatment 44

37 Hypothesis: ZD promotes carcinogenesis by activating cancer pathways not inhibited by genetic Cox-2 ablation Approach: 1. Transcriptome profiling of forestomach mucosa from ZD:Cox- 2 -/-, ZS:Cox-2 -/-, ZD:WT, and ZS:WT mice by Affymetrix GeneChip mouse genome array. 2. DAVID (Database for Annotation, Visualization and Integrated Discovery) bioinformatics to identify relevant biological processes/functions from expression data captured by transcriptome analysis 3. Ingenuity Pathway Analysis software (IPA) to analyze probable network/pathway and functional group enrichment 4. Investigation of identified markers in ZD:Cox-2 -/- mouse models of oral-esophageal cancers 5. Reconstitution studies to show that ZR attenuates inflammation and restores the antitumor effect of COX-2 blockade 45

38 Purpose: to determine if a ZD diet eliminates the antitumor effect of genetic Cox-2 disruption in NQOinduced tongue carcinogenesis as it does in NMBA-induced forestomach cancer Weanling mice were given drinking water exposure to NQO for 26 wks (20 ppm for 19 wks and 30 ppm for 9 wks). Wan et al., Int J Cancer Sep 20 46

39 Test the hypothesis that ZD promotes carcinogenesis by activating cancer pathways not inhibited by genetic Cox-2 ablation, by performing transcriptome profiling of forestomach from ZD:Cox-2 -/-, ZS:Cox-2 -/-, ZD:WT, and ZS:WT mice (n = 4/group). ZD:Cox-2 -/- ZS:Cox-2 -/- PCNA H&E 47

40 Summary of gene expression profiling data ZD causes extensive and high levels of gene expression changes in Cox-2 -/- mice than WT mice (P 0.05; fold-change=2) ZD:Cox-2 -/- vs ZS:Cox-2 -/- comparison: 314 dysregulated genes ZD:WT vs ZS:WT comparison: 67 dysregulated genes Cox-2 deletion causes limited expression changes in ZS forestomach (P 0.05; fold-change=2) ZD:Cox-2 -/- vs ZD:WT comparison: 90 dysregulated genes ZS:Cox-2 -/- vs ZS:WT comparison: 17 dysregulated genes 48

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43 IPA identifies a NF-kB centric network in ZD:COX-2 -/- vs ZD:COX-2 -/- forestomach of 35 genes, with 60% of the genes from the upregulated genes that included S100A8, thus predicting the activation of a S100A8 - NF-kB inflammatory pathway 51

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45 Investigate 1) if during malignant tongue/forestomach tumor progression S100A8 inflammation is In fact activated, and 2) if these carcinomas overexpress PCNA and p53, two prognostic factors in human oral cancer. 53

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47 Conclusions 1. The data show that with COX-2 pathway blockade prolonged dietary ZD causes chronic inflammation in the tongue/forestomach by activating alternative inflammatory RAGE-S100A8/A9 and p53 response pathways, thereby fueling tumor progression and bypassing the antitumor effect of Cox-2 deletion. 2. The data provide a likely mechanism to explain the inefficacy of such targeted cancer therapy in oral-cancer patients, since many of these patients are frequently Zn-deficient. 3. The finding that Zn-replenishment attenuates the inflammatory response and restores the antitumor effect of COX-2 blockade has important clinical implications. 4. Thus, stratification of patients by Zn status would be useful, and a personalized cancer therapeutic paradigm that includes Zn may improve efficacy. 55

48 Acknowledgements Past/Present Lab Members Yubao Jiang Shao-Gui Wan Honping Chen Ji-Xia Li Kin-Mang Lau Vu T. Ngyuen Liang Zhang Maurisa Riley Xianglan Liu Lauren Kauffman Collaborators TJU John L. Farber Karl J. Smalley OSU Carlo M. Croce Cristian Taccioli Hansjuerg Alder Kun Huang Chang-Gong Liu (MD Anderson) Funding: AICR ( ); NIH (CA118560) 56