Immunity Depletion, Telomere Imbalance, and Cancer-associated Metabolism Pathway Aberrations in Intestinal Mucosa upon Caloric Restriction

Systematic analysis of calorie restriction (CR) mechanisms and pathways in cancer biology has not been carried out, leaving therapeutic benefits unclear. Using a systems biology approach and metadata analysis, we studied gene expression changes in the response of normal mouse duodenum mucosa (DM) to short-term (2-weeks) 25% CR as a biological model. We found a high similarity of gene expression profiles in human and mouse DM tissues. Surprisingly, 26% of the 467 CR responding differential expressed genes (DEGs) in mice consist of cancer-associated genes—most never studied in CR contexts. The DEGs were enriched with over-expressed cell cycle, oncogenes, and metabolic reprogramming pathways (MRP) that determine tissue-specific tumorigenesis, cancer, and stem cell activation; tumor suppressors and apoptosis genes were under-expressed. DEG enrichments suggest a misbalance in telomere maintenance and activation of metabolic pathways playing dual (anti-cancer and pro-oncogenic) roles. Immune system genes (ISGs) consist of 37% of the total DEGs; the majority of ISGs are suppressed, including cell-autonomous immunity and tumor immune evasion controls. Thus, CR induces MRP suppressing multiple immune mechanics and activating oncogenic pathways, potentially driving pre-malignant and cancer states. These findings change the paradigm regarding the anti-cancer role of CR and may initiate specific treatment target development.


Introduction
Calorie restriction (CR), where test animals receive a reduced energy diet, is one of the most broadly acting regimens for preventing or reversing weight gain and inhibiting cancer in experimental tumor models (1,2). Protocols typically involve 10 to 40% reductions in total energy intake compared to ad libitum-fed controls, but with adequate nutrition and a controlled physical environment (2). Chronic reduction of dietary energy intake without malnutrition decreases adiposity, inflammation and improves metabolic profiles (3,4). CR was shown to increase tumor latency and have protective effects in some experimental mammary carcinogenesis models (5,6). Upon CR, metabolic alterations foster health-promoting characteristics including increased insulin sensitivity, decreased blood glucose and growth factors (IGF-1), and angiogenesis (4). Reducing IGF-1 and glucose levels may decrease tumor progression (2)(3)(4)7). Large bodies of experimental preclinical models support CR as anti-tumorigenic (2)(3)(4)7). Yet, data has also demonstrated neutral and negative effects (8)(9)(10). For instance, mice small intestinal response had a highly-dispersal trend decreasing large polyps (> 2mm) but increasing small polyp (≤2 mm) numbers (8). CR started early in life reduced incidence/delayed progression in most rodent tumors, however, CR started in middle-aged mice had higher lifetime incidences of lymphatic neoplasms (10). Whether CR results in protective or deleterious effects on cancer risk and outcome depends on length and restriction severity (6,7). A dominating anti-cancer CR effect decreases tumor rate growth via inhibition of circulating systemic factors (e.g., hormones/growth factors) which stimulate cancer cell proliferation (5,11). However, many cancer subtypes lack hormone/growth factor sensitive cells (e.g., high aggressive basal-like cancers). Sensitive tumor clone(s) may be targets of prooncogenic metabolic reprogramming and/or replaced with more aggressive CR-resistant ones.
Recent randomized clinical trials have been explored for potential CR anti-cancer properties (12)(13)(14). These trials showed mixture effects of CR directly on tumor tissue growth, host immune cells response, and other tissue responses (e.g., adipocytes). However, these studies do not mechanistically support the anti-cancer role of CR on neoplastic processes as indicated by large bodies of empirical experimental model data. CR-mediated reduction in cancer cell proliferation is central to anti-cancer animal model studies. The clinical trial studies showed no negative energy/calorie restriction effects on cell proliferation marker Ki-67 in Barrett's esophagus (13) and breast cancers (14). In men with prostate cancer, presurgical weight loss showed CR-mediated Ki-67 upregulation (12). Additionally, high overexpression of several periodic cell cycle genes, cancer-associated genes, and oncogenes have been found (12,14).
The duodenum mucosa (DM) is a useful biological model for the study of CR small intestine response (15). After epithelial cells, the most numerous cells in the lamina propria are immune cells, mainly duodenal intraepithelial lymphocytes (IEL) (16,17), present at 9-50 IELs per 100 epithelial cells that vary in different medical and bacterial conditions (17,18). The DM transcriptome model showed dichotomized differentially expressed genes (DEGs) response to CR, with metabolic genes upregulated and immune/inflammatory genes downregulated (15). However, DM tissue-specific transcriptional network profiles in CR-medicated cancer biology mechanics were not studied. The intestinal mucosa epithelium is the most highly proliferative mammalian tissue and CR further enhances epithelial regeneration (19,20). Precise balances control the quiescent (G0-phase) and active intestinal stem cells, progenitors, and stroma cells. In healthy mice, short-term CR reduced cellular mass resulting in 15% shorter villi, reduced numbers of more differentiated epithelial progenitor cells, while the proliferative rate and the number of Lgr5 (high)/Olfm4+ active intestinal stem cells were modestly increased (21). Isolated crypts from CRrestricted mice in vitro can form primary and secondary organoid bodies with increased proliferation rate and stem cell growth per crypt (21). Cycling intestinal stem cells exhibit high Wnt activity that sensitizes them to DNA damage after CR (21,22). This suggests CR initiates microenvironmental/stroma-mediated control loss, activates independent proliferation, and stimulates progression of stem cell organoid body formation-cancer hallmark factors.
The direct CR anti-cancer and pro-cancer mechanics in normally proliferated intestinal mucosa are poorly understood. Systematic analysis of CR mechanisms in cancer biology has not been carried out, making therapeutic benefits unclear (12,23,24). The contradictive tumor biology CR data motivated us to use hypothesis-testing and data-driven system biology analysis of gene expression profiles changes in small intestine mucosa tissue upon short-term CR.
Our major objective is to identify cancer driver genes and oncogenic pathways induced by CR-related perturbation of cellular homeostasis affecting normal proliferation of epithelium mucosa via metabolic reprogramming and depletion of immune system control. Our central hypothesis is that CR activates metabolic reprograming pathways in DM defined by cancer hallmarks (e.g., uncontrolled cell proliferation, tumor suppressors loss etc.), while suppressing immune system surveillance; this may initiate occurrence or preferential competition of abnormal proliferating cells leading to pre-cancer and cancer risks. To test this hypothesis, we developed unbiased and comprehensive data analysis approaches. We use the mouse mucosa response to 2week 25% CR model in DM. Our results demonstrated CR induces drastic gene expression and pathway suppression of intracellular immunity and immune responses of T-, B-, NK-cells, macrophages and their precursors. We identify and characterize CR DEGs specifying regulatory networks modulating telomere stability and tumor suppressors, activating proliferative tissuespecific cancer-like stem cells, oncogenes, and chemical carcinogenesis. New CR response genes, perspective treatment onco-targets, and cancer risk factors are discussed. Our CR-induced metabolic reprogramming and multi-cellular competition models suggest plausible dysregulation of genes, networks, and pathways of pre-malignant and malignant states.

Materials and methods
In this study, we carried out data analysis of differentially expressed genes (DEGs) of 26,966 probe sets (p.s.) from Affymetrix MoGene 1.0 ST microarray data from mouse mucosa scrapings produced in our previous study (15). Using statistical criteria reported in (15), 521 p.s. were selected. 16 p.s without gene annotations were excluded. We determined 505 probe sets (p.s.) representing CR mice DEGs, with 467 unique annotated gene symbols (Supplementary Data 1A-v11 determined the functional associations between the human and mouse orthologous genes. Figure 1B and Supplementary Table S1 present the top enriched GO terms and pathways for the 382 CR DEGs with human orthologs. Gene set enrichment analysis (GSEA) indicated a dichotomization response in mouse CR microarray gene expression (metabolic and inflammatory) which we previously confirmed by qPCR (15) ( Figure 1C).

Telomeric maintenance pathways and cell cycle gene responses in CR mice
Mice mucosa cellular immune system compartments are deeply suppressed by CR and epithelial network interactions are activated Upon CR, strong/global immune system suppression and epithelial activation occurs. The 171 ISGs consist 37% (171/467) of CR-responding DEGs. Stat1 (immune) and Ppara (metabolic) drivers induce de novo abnormal regulation of DM pathways. The immune and epithelial network (Figure 3) contained 118 genes, 311 edges (protein interactions), average number of neighbors 3.64, clustering coefficient 0.14, network density 0.02, and PPI network enrichment p < 1.00 10 -16 . Subcellular ISG and ECG localization networks are in Supplementary Figure S1. The suppressed ISGs were characterized using the Disease and Biological Function annotation tool from IPA (Supplementary Table S3). The Lymphoid Tissue Structure and Development categorization (p-value dynamical range: 4.07 10 -19 to 2.94 10 -3 ) included 66 genes within its categories. The functional annotations proliferation of B lymphocytes (p = 9.52 10 -10 , n = 18), T cell development (p = 5.12 10 -9 , n = 24), development of antigen presenting cells (p = 7.18 10 -7 , n = 10), and NK cell proliferation (p = 7.31 10 -6 , n = 8), were displayed as a network with 31 genes ( Figure 4A).
Additionally, 45 T cell-associated functional annotations ( Figure 4B) and 27 B cell associated functional annotations were displayed ( Figure 4C). Sixty Inflammatory Response genes (p-value dynamical range: 2.47 10 -16 to 2.94 10 -3 , n = 77) were selected ( Figure 4D). An additional suppressed ISG functional annotation network is provided in Supplementary Figure S2. Our analysis determined a deep suppression of cellular immune system genes belonging to all tissue-associated lymphocyte populations and immune system regulatory cells, suggesting systemic immune-cell-specific quantity reduction in the mucosa.
CR induces downregulation of embryonal/haematopoiesis/immune CSC genes, however upregulation of epithelial cell CSC genes Cancer stem cells (CSCs) are a subpopulation of cancer cells possessing characteristics associated with normal stem cells, specifically self-renewal and differentiation giving rise to the cell types found in a particular cancer sample (38
The immune system, cancer-associated, epithelial, and telomere subsets were displayed as a network with the Sirtuin Signaling Pathway overlaid (Figure 6). The network has 132 connected components, 227 non-connected components, 350 edges, average number of neighbors 3.74, clustering coefficient 0.14, network density 0.02, and PPI network enrichment p < 1.00 10 -16 . Supplementary Figure S3 shows the network interactions of the tumor-immune and tumorepithelial microenvironments identifying possible mechanisms of CR-induced malignancy risks or anti-cancerogenic effects.

Interferon-inducible DNA-binding gene family members on Chr 11 are CR-suppressed
One of the top CR DEGs, predicted/uncharacterized protein-coding gene 5431 (Gm5431) localized in Chr 11qB(1.2), was strongly suppressed (FC =-6.76; p = 2.7 10 -17 ). We observed that the gene belongs to Chr11qB(1.2) locus containing a family of paralog genes that encode interferon (IFN)inducible GTPase proteins, also called immunity-related p47 GTPases (INF-I GTPases). Interferons induce intra-cellular programs functional in innate and adaptive immunity against infectious pathogens. Chr 11qB(1.2) locus comprises Gm5431 with genes Irgm1, Tgtp1, Tgtp2, Ifi47 and uncharacterized genes 9930111J21Rik1, 9930111J21Rik2, Gm12185, Gm12186 and Gm12187 (and their transcribed isoforms) ( Figure 8A, Supplementary Data 5). Nine of the ten genes are localized on the chromosome negative strand. Genes in this locus have high evolutionarily conserved sequences and paralogous in several other loci (for instances, Chr 11qB(1.3) (Irgm2, Igtp), Chr18 (Iigp1, F830016B08Rik)), in other chromosomes and orthologous in genomes of many species ( Figure 8A, Supplementary Figure S4). IRGM in humans is an ortholog to this family, with links to Chron's disease, inflammatory disorders, and non-alcoholic fatty liver disease (42). Irgc1 (with RefSeq transcript NM_199013) located on Chr 7 is also an ortholog of the gene family to the human genome (IRGC) but its transcription was not significantly regulated by CR (Supplementary Figure S4).
Using MAFFT-DASH -multiple alignment software integrated with structural search (43), we found high confidence similar sequences and domains between IIGP1 and all our proteins (Supplementary Data 5). Longer proteins encoded by Gm5431, 9930111J21Rik1, and 9930111J21Rik2 show the highest sequence similarity with IIGP1/1TQ4 and include two nonoverlapping IIGP1 sequence homologous. IIGP1, the member of INF-I GTPases encoded by gene Iigp1 localized on Chr18, has known crystal structure and atom resolution 3D model 1TQ4. The IIGP1/1TQ4 is built of two domains, the G domain and a helical domain including five DNAbinding motifs (44). Due to high sequence similarity and presence of evolutionarily conserved sequence domains (Figure 8A), we suggest structural and functional similarity of IIGP1 and the members of the INF-I GTPase family analyzed in this section.
The transcription levels of the IFN-I-GTPases were commonly suppressed due to CR response ( Figure 8B). Furthermore, the Chr. 11qB(1.2) loci is flanked by CTCF binding sites. Analysis of the IFN-inducible gene family members suggests that CR in DM induces global suppression of interferon-induced cellular functions in innate and adaptive immunity.

Drug targets in energy-responsive metabolic pathways
A total of 36 drug targets were associated with the 467 mouse duodenum CR DEGs as identified by IPA (Supplementary Data 6). PPARa, NOS2, TLR4, and CXCL10 are the most relevant strongest hubs with the highest connectivity determined by network analysis. TLR4 is targetable by eritoran, GSK1795091, OM 174 lipid, and resatorvid. The activity of CXCL10 is modulated by MDX-1100. Anti-CXCL10 monoclonal antibodies have implications in infectious disease, chronic inflammatory, and autoimmune disease therapeutics and attenuate murine inflammatory bowel disease and murine AIDS colitis (45). Agents that deplete cellular glutathione metabolism in combination with arsenic trioxide are studied for the treatment of non-acute promyelocytic leukemia (46). Attempts to increase the efficacy of cancer treatments via lifestyle or diet changes would benefit from further examination of these top targets in nutrient-responsive pathways.

Discussion
Because pre-malignant and cancer cells must reprogram their metabolic state in every step of progression to survive in nutrient-altered conditions, metabolic reprogramming is recognized as a cancer hallmark (47). In our study, we for the first time identified cancer driver genes and genes playing key roles in oncogenic pathways induced by CR perturbation of cellular composition, tissue homeostasis and metabolic reprogramming, leading to cell proliferation of epithelium mucosa and global depletion of immune system control.
We found that 26% of the CR responding differential expressed genes (DEGs) in mice DM consist of cancer-associated genes-most never studied in CR contexts. These responses may lead to perturbation of telomere maintaining mechanisms, and activation of the cell cycle, prooncogenic, and stem-like cancer cell pathways including EMT. CR-induces metabolic reprogramming, which affects the ISGs, consisting of 37% of the total DEGs; the majority of ISGs are suppressed, including cell-autonomous immunity and tumor immune evasion controls.
Major findings of this study: 1) CR induces tissue homeostasis dysregulation, shifting the transcriptome profile to pro-oncogenomic pathway patterns in DM associated with activation of metabolism and proliferative activity of epithelial cells, but depletion of intracellular immunity and functions of all immune cell types. 2) CR induces transcription of cell cycle genes including a subset of key cancer-associated signaling genes directly involved in reprogramming pathways, premalignancy, malignancy states, and poor outcome in mice and humans. 3) In CR response, apoptotic gene expression is reduced or not significantly varied. 4) Tissue-specific proliferative epithelial stem DEGs are activated, however, immune-specific stem cell progenitor genes are suppressed upon CR. 5) CR induces transcription activation of key tumor-susceptible and oncogenes gene sets and their networks with tissue-specific risk of carcinogenesis and downregulates protective mechanisms mediated by key tumor suppressor genes. 6) CR induces multiple transcription suppression effects in autophagy, tumor-immune surveillance mechanics and genes associated with induction and effector stages of NK, T-, B-cells and macrophages immune response. 7) Detoxifying exogenous chemicals enzymes and drug metabolism networks with glutathione pathways are activated upon CR, but could be involved in anti-cancer and pro-cancer outcomes via mutagenesis and DNA damage/repair mechanisms.
Our network and pathway analysis provide interconnections of CR DEGs with biological functions, molecular mechanisms, cell types and cellular compartments. Figure 9 shows the two-compartmental response models which hypothesise possible dysregulation mechanics of tissue/cell homeostasis upon CR in intestinal mucosa tissue leading to pro-oncogene pathway activation and increased potential risk of premalignant and cancer states. Supplementary Figure  S5 shows key pathways and gene targets that may be involved in the multiple disbalances increasing risk of metabolic reprogramming leading to malignant states. Table 1 shows the selected list of the CR-response genes that expression is strongly supported by literature data as key genes of oncogenesis, immune suppression, and metabolic reprogramming and could be considered and tested as CR-induced cancer risk markers. According to our model, long-term CR may confer increased tumorigenic risks if there are durable and sustained increases in stem mutations and abnormal proliferation, DNA damage, epigenetic modification, and cell numbers that can undergo mutagenesis. Activated stem cells of DM villi may exhibit reprogramming behavior if metabolic machinery is used at higher rates, in combination with aggressive chemical and pathogenic bacterial factors. While external to our analysis, bacteria (H. Pylori), parasites, and viruses may provide the mutagenesis factors initiating oncogenesis in mucosa epithelial cells. Pathogenic microbiota signals could also act on the intestinal stem cell niche and homeostasis upon CR (48).
The multiple gene dysregulation in mucosa upon CR response could be interpreted as an "early ischemia"-like syndrome observed in newborns, demonstrating marked reduction of villous height and increasing epithelium crypt-villus ratio (in the vertical axis) in combination with small numbers of immune cells in intestinal lymphoid structures along the lamina propria (horizontal axis) (49). These changes may resemble those seen in autolysis. Reduction or no change in apoptosis gene expression in epithelial cells occurs and no acute inflammatory cells are activated (49). This coincides with global INF-induced autophagy reduction in our study.
CR induces epithelial cell cycle proliferation and suppresses apoptosis, increasing cancer risk. We observed suppressed apoptotic genes upon CR. Fasting, short-term, and long-term dietary restriction almost uniformly reduces cellular proliferation (liver, bladder, skin, heart, colorectum) explaining anticarcinogenic effects of CR (5). A potential pro-cancerogenic effect in fasted-refed animals resulted from proliferation increases with apoptosis decreases in response to refeeding (5). CR in some experimental models enhances cell death rate, however, proliferation/ apoptosis rates vary through the course of treatment (5,11,21). CR enhances the proliferation of Lgr5+ intestinal stem cells, the cell-of-origin for intestinal precancerous adenomas and leads to the first organoid and acceleration of the second organoid formations (11,21).
In normal physiological conditions, cell cycle and apoptosis gene activity in tissue-specific stem cells, progenitor, and differentiated epithelial cells maintains tissue homeostasis. However, short-term CR disturbs this balance and may switch-on pro-oncogenic pathways. For instance, Rrm2 is high upregulated in DM CR response. RRM2 is an oncogene playing a key role in tumorigenesis and cancer progression, including colorectal and oesophageal cancers (oncoMX.org) (50)(51)(52)(53)(54). CR-activated cell cycle periodic genes in our study are directly or indirectly involved in tumorigenesis pathways, drive cancer cell proliferation and aggressiveness (RRM2, ACSL3, RBBP8, KMO, FKBP5), drug resistance (ABCC5) and modulate chemosensitivity (FKBP5) (references in Supplementary Table S2A). Fkbp5 is the most over-expressed CRresponse gene. This androgen-responsive gene has high expression in esophageal adenocarcinoma (EAC) tissues and is associated with decreased patient survival (55). FK506-binding protein 5 (FKBP5) plays a pro-oncogenic role in EAC (55,56). In tissue resection specimens from EAC patients, FKBP5 expression was positively associated with proliferation as measured by Ki-67 expression, which was also observed in the prostate cancer weight loss clinical trials (12,56). FKBP5 is a cis-trans prolyl isomerase that binds to the immunosuppressants FK506 and rapamycin. The number of activated CR mice cell cycle DEGs and their expression level increments (fold change) were stronger in the upregulated genes vs down-regulated DEGs. Our model showed high upregulation of FKBP5 and RRM2 (FC=8.5 and FC=4.0 respectively) compared to downregulated TGM2 and PAQR4 (FC=3.1 and FC=1.76 respectively). Tgm2 and Paqr4 may provide suppressive effects in some aggressive cancer cells (Supplementary Table  S2B) (57,58). TGM2-siRNA knockdown attenuated colorectal cancer cell growth through the wnt3a/β-catenin/cyclin D1 pathway (58).
Our model includes potential gene regulatory switches modulating anti-cancer to prooncogenic CR response. CR induces specific mechanisms involved in pathogenic or adaptative EMT-driven stem cell response and proliferation of mucosa epithelial cells. The EMT pathway gene expression alteration (36) may or may not indicate markers solely, but silencing/dormant states could be formed. Due to CR-induced immune system depletion and mutagenesis/genotoxic events (leading to occurrence and accumulation of a driver mutation and genome instability), the risks of oncogene activation and tumor suppressor depletion required for conversion to tumorinitiating cells over long periods could increase (60). This suggests that tumor lesions may need to be studied in experimental systems over time with lower threshold detection level of small benign polyps and dormant malignant states (8). Time course systems biology analyses of CR severity and length should be conducted on pre-existing tumor dormant states (5,11).
Oncogenes and pro-oncogenic genes are poorly studied in CR and carcinogenesis contexts. Searching in PubMed revealed only 32.2% (39/121) of our CR cancer-associated genes (Supplementary Data 7) are associated with terms "caloric" or "calorie" or "caloric restriction" or "calorie restriction" or "dietary restriction". Supplementary Table S6 includes upregulated oncogenes upon CR: Cemip, Tns4, Aldh1a1, Rab30, Rrm2, and Gsta3. CEMIP mRNA overexpression correlates with poorer colon cancer patient survival and facilitates colorectal and stomach tumor growth (OncoMX) (24). TNS4 expression is transcriptionally regulated by MAP kinase signaling pathway and plays a critical role in tumorigenesis in several tissues including the colon. TNS4 and ALDH3A1 expression levels were increased in HCT-8 colon cancer cells and influence cancer cell migration, invasion, and proliferation (61). RAS oncogene family member 30 (RAB30) was upregulated in the microarray of epithelial colorectal adenocarcinoma and acute lymphoblastic leukemia-derived cell lines (62). PPARα-sensitive genes during starvation include Cxcl10 and Rab30 (63). RRM2 plays oncogenic roles in tumorigenesis (50) and is a poor prognostic factor for colon, breast, and pancreatic cancers (51)(52)(53)(54).
Glutathione (GSH) plays a dual role in cancer progression. The Glutathione S-transferases (GSTs), phase II detoxification enzymes, were activated in CR mice: membrane-bound microsomal (Mgst1, Mgst2) and cytosolic family members (Gsta1, Gsta2, Gsta3, Gsta4, Gstm1,  Gstm2, Gstm3, Gstm4, Gstm6, Gstk1). In healthy cells, it is crucial for removal and detoxification of carcinogens (64). However, GSH metabolism is associated with colorectal cancer pathogenesis (65). The GSH system regulates proliferation and survival by offering redox stability in a variety of cancers (66,67). MGST1 is crucial for stem differentiation (68). MGST1 polymorphisms may contribute to CRC risk (69). GST-overexpressing phenotypes are present in many drug-resistant tumors (including breast, colon, and lung cancers) (66). We propose therapies targeting the GSH antioxidant system in tumors in combination with CR may sensitize cancer cells to treatment (64) and reduce the risk of cancerous effects in CR conditions. Mice treated with L-buthioninesulfoximine depleted GSH levels in esophageal cancer and decreased tumor burden, inhibited cell proliferation, and activated cell apoptosis (70).
The Sirtuin Signaling Pathway mediates CR-responded cancer-associated networks. This pathway regulates cancer cell metabolic reprogramming in glucose-poor environments (71). Pck1, a CR upregulated Sirtuin Signaling Pathway gene, is associated with cancer cell gluconeogenesis and increased PCK1 expression is crucial for cancer growth in the absence of glucose (72). In our study, upon CR in mice DM, expression of Sirt3 isoforms (probe set 10568997, NM_022433) was downregulated. Sirtuin 3 (SIRT3) is a major NAD(+)-dependent mitochondrial deacetylase and the key regulator of fundamental processes frequently dysregulated in aging and other diseases (40,41). New experimental designs may be pivotal to validate SIRT3 as a cancer regulator in CR.
CR induces multiple transcription suppression effects in the genes of autophagy and tumor immune surveillance mechanics. In contrast to activation of the metabolic genes associated with epithelial cells, we found immune system transcription suppression in 37% of DEGs induced by CR. Restricted cell cycle genes in interferon-inducible pathways include antiviral and anti-cancer activity (73) (Ifit1 and Ifit2) and pro-inflammatory reactions recruiting immune cells to target cells (74) (genes Stat1 and Unc5cl) (Supplementary Table S2B).
Negative role of CR in tumor-immune surveillance. The immune system classified CRresponding DEGs were majorly suppressed (Supplementary Table S10). An intact immune system is essential to prevent neoplastic cell development and progression (75,76). Mice with a homozygous deletion of the Rag-2 alleles completely lack NK-like T, T and B cells and have increased incidence and growth of spontaneous tumors and chemically induced cancer lesions (77). IFN-g forms the basis of an extrinsic tumor-suppressor mechanism in immunocompetent hosts (75,76,78). STAT1 can suppress tumor formation (75). The elimination of interferon IFN-g or STAT1 genes in mice resulted in increased incidence and growth of spontaneous and chemically induced tumors (75,77,78). CR may impact IFN-mediated tumor elimination (79).

CR induces suppression of INF pathways in DM.
Type II interferon signaling (IFN-g) is suppressed by CR. IFN-inducible GTPases play central roles in defending the mammalian cell's interior against a diverse group of invading pathogens. The IFN-inducible GTPases ability to control infection at the level of an individual cell-a process termed cell-autonomous immunityis responsible for microbial killing (80). We identified IFN-inducible GTPase genes on Chr11q B(1.2) which were downregulated upon CR. Ifit1, Ifit2, Ifit3, Ifi203, Ifi44, Ifi27l2a, and Irf1 (all CR-suppressed) function in antiviral and antimicrobial response. Ifit1 knockout promoted viral replication in murine norovirus infected cells (73). Loss of interferon regulatory factor 1 (IRF1) function causes severe susceptibility to infections in mice and humans (81). IRF1 suppresses tumor cell growth, stimulating immune responses against tumor cells (81)(82)(83)(84). Defects in IRF1 are associated with gastric and lung cancer, and myelogenous leukemia (82). The Stat1/2 and Irf1/9 CR-network interactions mediate immunity and gut inflammation (74).
In summary, our results suggest that CR-mediated metabolic reprogramming suppresses multiple host tumor surveillance prevention mechanics including cell-autonomous immunity and activates signaling pathways of pro-oncogenes, tissue-specific cycling and silenced stem cells, and cancer predisposition genes driving pre-malignant and cancer states. This study's limitations refer to meta-data sets available for analysis, data type (e.g., microarrays), DM expression profile composition (cellular mixture), CR experimental time design, and pre-clinical models of CR effects on tumor pathobiology. However, our systems biology, data-driven, and hypothesis-testing approach to metadata provides feasibility by identifying DM DEGs subsets according to cell-type specificity functions: IFN-inducible GTPases genes, CR-induced cancer-and immune system cross-talks. This provides multiple determinants of aberrant signalling, cell-cell interaction networks leading dynamically to cell transformation, clonal selection, immune response delay, immune surveillance suppression and tumorigenesis (85).
These findings may change the paradigm regarding the anti-cancer role of CR and initiate new unbiased CR cancer biology studies. Clearly, no single cell type/subtype or unique pathway accounts for all anti-cancer or pro-oncogenic effects of CR in normal, but complex mucosa tissue. As with most chronic disease intervention strategies, combination approaches improving lifestyle (CR, diet, physical activity), prophylactic strategies and reproducible pharmacological interventions that target specific cells and their pathways are needed to prevent pre-cancer and cancer states. Future (bioengineering) directions to implement our results is to identify novel drugs and CR mimetics, compounds that mimic the specific CR mechanism. This will allow protection against cancerous metabolic tissue reprogramming pathways, chromosome stability, provide normal stem cell differentiation and proliferation, ensure physiologic cellular composition balances in target the tissue, and protect the immune system from suppression or hyper-activation.
Our computationally predicted approaches provide new platforms and resources for the formulation and analysis of testable hypothesis for CR in pro-cancer and cancer prevention mechanisms. Our models could be useful for further modelling and experimental validations of CR-response protecting metabolic reprogramming pathways. Further study is encouraging for the perspective utilization of CR-associated mechanisms in clinical oncology strategies.

Conclusion:
• CR dramatically reduces immune responses, dysregulates tissue-specific epithelial stem cell developmental processes and telomere maintaining processes • CR induces metabolic reprogramming processes, oncogenic and cell cycle pathways, and may increase the risk of malignancy • CR-induced Rrm2, Lamc2, Fkbp5, and aberrant glutathione gene family activation coupled with Sirtuin3 and RNaseL suppression could play tumorigenic roles in mucosa pathophysiology • Interferon-inducible gene family including novel members on Chr11qB1.2 are suppressed by CR

Data Availability
The microarray datasets analyzed during the current study are available in the ArrayExpress repository, https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-6248.              CR induces network between DEGs of glutathione metabolism (purple; mmu00480), chemical carcinogenesis (green; mmu05204), and sirtuin signaling pathways (magenta). Sirtuin signaling regulation has downstream targets in glutathione metabolism (Gstk1, Gsta4, Mgst1, Gstm6) and chemical carcinogenesis (Cyp2b10, Cyp3a44). Black lines indicate molecule relationships. Self-loops were removed for easier visualization of network interactions between molecules. Asterisks on molecules indicates druggable targets: Ppara (aleglitazar, atorvastatin/choline fenofibrate, bezafibrate, clofibrate, docosahexaenoic acid, ezetimibe/fenofibrate/simvastatin, gemfibrozil, NS-220, pemafibrate, TPST-1120, tesaglitazar), Nos2 (GW 273629, N(G)-monomethyl-D-arginine, pimagedine, triflusal), Cyp3a44 (atazanavir/cobicistat/darunavir), Abca1 (probucol), Gss (N-acetyl-L-cysteine), Pfkfb3 (PFK-158). All genes are adjusted p-value < 0.05 at |FC| > 1.5, except Sirt3 with FC-1.38.    . CR cellular disbalance models through immune and autophagy suppression. CRinduced metabolic reprogramming and immune suppression. DEGs and pathways analyses strongly support our hypothesis that CR dramatically changes the epithelial versus immune cells relationships. Functional interactions modulating the homeostasis cooperation between cell types may increase malignancy risk. Morphologically, mucosa enterocyte differentiation and their cell type density depends on the cell position along both the vertical axis (crypt-villus) and horizontal axis (proximal to distal) of the GI tract. At steady-state in normal conditions (homeostasis) of mucosa tissue, the cell types at differentiation states are position-dependent and relationships between cell types remains balanced. Our results suggest that CR induces a hypocellular and multiple cell-types disbalance by stimulation of cell proliferation and autonomation of this process. Our model suggests that CR response leads to metabolic activation, inducing cell cycle genes and proliferation of epithelial stem cells. CR reduces the total mass of epithelial cells (vertical axis) (21), autophagy, apoptosis mechanisms, and telomere stability. Immune cell populations including progenitors and effector cells are globally reduced, suggesting increased pathogen susceptibility. If the processes controlling DNA and RNA damage induced by chemical carcinogens (49,64) become dysregulated, it increases the risk of transformation and uncontrolled proliferation of abnormal stem-like cells.