Combined inhibition of MTAP and MAT2a mimics synthetic lethality in tumor models via PRMT5 inhibition.

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.

The nutritional environment determines which and how intestinal stem cells contribute to homeostasis and tumorigenesis.

Sporadic colon cancer accounts for approximately 80% of colorectal cancer (CRC) with high incidence in Western societies strongly linked to long-term dietary patterns. A unique mouse model for sporadic CRC results from feeding a purified rodent Western-style diet (NWD1) recapitulating intake for the mouse of common nutrient risk factors each at its level consumed in higher risk Western populations. This causes sporadic large and small intestinal tumors in wild-type mice at an incidence and frequency similar to that in humans. NWD1 perturbs intestinal cell maturation and Wnt signaling throughout villi and colonic crypts and decreases mouse Lgr5hi intestinal stem cell contribution to homeostasis and tumor development. Here we establish that NWD1 transcriptionally reprograms Lgr5hi cells, and that nutrients are interactive in reprogramming. Furthermore, the DNA mismatch repair pathway is elevated in Lgr5hi cells by lower vitamin D3 and/or calcium in NWD1, paralleled by reduced accumulation of relevant somatic mutations detected by single-cell exome sequencing. In compensation, NWD1 also reprograms Bmi1+ cells to function and persist as stem-like cells in mucosal homeostasis and tumor development. The data establish the key role of the nutrient environment in defining the contribution of two different stem cell populations to both mucosal homeostasis and tumorigenesis. This raises important questions regarding impact of variable human diets on which and how stem cell populations function in the human mucosa and give rise to tumors. Moreover, major differences reported in turnover of human and mouse crypt base stem cells may be linked to their very different nutrient exposures.

Environmental Impact on Intestinal Stem Cell Functions in Mucosal Homeostasis and Tumorigenesis.

Multiple cell compartments at or near the base of the intestinal crypt have been identified as contributing intestinal stem cells for homeostasis of the rapidly turning over intestinal mucosa and cells that can initiate tumor development upon appropriate genetic changes. There is a strong literature establishing the importance of the frequently dividing Lgr5+ crypt base columnar cells as the fundamental cell in providing these stem cell-associated functions, but there are also clear data that more quiescent cells from other compartments can be mobilized to provide these stem cell functions upon compromise of Lgr5+ cells. We review the data that vitamin D, a pleiotropic hormone, is essential for Lgr5 stem cell functions by signaling through the vitamin D receptor. Moreover, we discuss the implications of this role of vitamin D and its impact on relatively long-lived stem cells in regards to the fact that virtually all the data on normal functioning of mouse Lgr5 stem cells is derived from mice exposed to vitamin D levels well above those that characterize the human population. Thus, there are still many questions regarding how dietary and environmental factors influence the complement of cells providing stem cell functions and the mechanisms by which this is determined, and the importance of this in human colorectal tumor development. J. Cell. Biochem. 118: 943-952, 2017. © 2016 Wiley Periodicals, Inc.

Vitamin D is a determinant of mouse intestinal Lgr5 stem cell functions.

Lgr5+ intestinal crypt base columnar cells function as stem cells whose progeny populate the villi, and Lgr5+ cells in which Apc is inactivated can give rise to tumors. Surprisingly, these Lgr5+ stem cell properties were abrogated by the lower dietary vitamin D and calcium in a semi-purified diet that promotes both genetically initiated and sporadic intestinal tumors. Inactivation of the vitamin D receptor in Lgr5+ cells established that compromise of Lgr5 stem cell function was a rapid, cell autonomous effect of signaling through the vitamin D receptor. The loss of Lgr5 stem cell function was associated with presence of Ki67 negative Lgr5+ cells at the crypt base. Therefore, vitamin D, a common nutrient and inducer of intestinal cell maturation, is an environmental factor that is a determinant of Lgr5+ stem cell functions in vivo. Since diets used in reports that establish and dissect mouse Lgr5+ stem cell activity likely provided vitamin D levels well above the range documented for human populations, the contribution of Lgr5+ cells to intestinal homeostasis and tumor formation in humans may be significantly more limited, and variable in the population, then suggested by published rodent studies.

The intestinal epithelial cell differentiation marker intestinal alkaline phosphatase (ALPi) is selectively induced by histone deacetylase inhibitors (HDACi) in colon cancer cells in a Kruppel-like factor 5 (KLF5)-dependent manner.

The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect.

Paneth cell marker expression in intestinal villi and colon crypts characterizes dietary induced risk for mouse sporadic intestinal cancer.

Nutritional and genetic risk factors for intestinal tumors are additive on mouse tumor phenotype, establishing that diet and genetic factors impact risk by distinct combinatorial mechanisms. In a mouse model of dietary-induced sporadic small and large intestinal cancer in WT mice in which tumor etiology, lag, incidence, and frequency reflect >90% of intestinal cancer in Western societies, dietary-induced risk altered gene expression profiles predominantly in villus cells of the histologically normal mucosa, in contrast to targeting of crypt cells by inheritance of an Apc(1638N) allele or homozygous inactivation of p21(Waf1/cip1), and profiles induced by each risk factor were distinct at the gene or functional group level. The dietary-induced changes in villus cells encompassed ectopic expression of Paneth cell markers (a lineage normally confined to the bottom of small intestinal crypts), elevated expression of the Wnt receptor Fzd5 and of EphB2 (genes necessary for Paneth cell differentiation and localization to the crypt bottom), and increased Wnt signaling in villus cells. Ectopic elevation of these markers was also present in the colon crypts, which are also sites of sporadic tumors in the nutritional model. Elevating dietary vitamin D(3) and calcium, which prevents tumor development, abrogated these changes in the villus and colon cells. Thus, common intestinal cancer driven by diet involves mechanisms of tumor development distinct from those mechanisms that cause tumors induced by the rare inheritance of a mutant adenomatous polyposis coli (Apc) allele. This is fundamental for understanding how common sporadic tumors arise and in evaluating relative risk in the population.

Intestinal stem cells: guardians of homeostasis in health and aging amid environmental challenges.

The intestinal epithelium is the first line of defense and acts as an interface between the vast microbial world within the gastrointestinal tract and the body's internal milieu. The intestinal epithelium not only facilitates nutrient absorption but also plays a key role in defending against pathogens and regulating the immune system. Central to maintaining a healthy epithelium are intestinal stem cells (ISCs), which are essential for replenishing the intestinal epithelium throughout an individual's lifespan. Recent research has unveiled the intricate interplay between ISCs and their niche, which includes various cell types, extracellular components, and signaling molecules. In this review, we delve into the most recent advances in ISC research, with a focus on the roles of ISCs in maintaining mucosal homeostasis and how ISC functionality is influenced by the niche environment. In this review, we explored the regulatory mechanisms that govern ISC behavior, emphasizing the dynamic adaptability of the intestinal epithelium in the face of various challenges. Understanding the intricate regulation of ISCs and the impact of aging and environmental factors is crucial for advancing our knowledge and developing translational approaches. Future studies should investigate the interactive effects of different risk factors on intestinal function and develop strategies for improving the regenerative capacity of the gut.

Non-stem cell lineages as an alternative origin of intestinal tumorigenesis in the context of inflammation.

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, has been shown to suppress intestinal stemness. Here, we used Paneth cells as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation in mice. Upon inflammation, Paneth cell-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in patients with inflammatory bowel disease, but also of a larger fraction of human sporadic colon cancers. The latter is possibly because of the inflammatory consequences of western-style dietary habits, a major colon cancer risk factor. Machine learning methods designed to predict the cell-of-origin of cancer from patient-derived tumor samples confirmed that, in a substantial fraction of sporadic cases, the origins of colon cancer reside in secretory lineages and not in stem cells.

EOGT enables residual Notch signaling in mouse intestinal cells lacking POFUT1.

Notch signaling determines cell fates in mouse intestine. Notch receptors contain multiple epidermal growth factor-like (EGF) repeats modified by O-glycans that regulate Notch signaling. Conditional deletion of protein O-fucosyltransferase 1 (Pofut1) substantially reduces Notch signaling and markedly perturbs lineage development in mouse intestine. However, mice with inactivated Pofut1 are viable, whereas complete elimination of Notch signaling in intestine is lethal. Here we investigate whether residual Notch signaling enabled by EGF-domain-specific O-linked N-acetylglucosamine transferase (Eogt) permits mice conditionally lacking Pofut1 in intestine to survive. Mice globally lacking Eogt alone were grossly unaffected in intestinal development. In contrast, mice lacking both Eogt and Pofut1 died at ~ 28 days after birth with greater loss of body weight, a greater increase in the number of goblet and Paneth cells, and greater downregulation of the Notch target gene Hes1, compared to Pofut1 deletion alone. These data reveal that both O-fucose and O-GlcNAc glycans are fundamental to Notch signaling in the intestine and provide new insights into roles for O-glycans in regulating Notch ligand binding. Finally, EOGT and O-GlcNAc glycans provide residual Notch signaling and support viability in mice lacking Pofut1 in the intestine.

Intestinal stem cell aging at single-cell resolution: Transcriptional perturbations alter cell developmental trajectory reversed by gerotherapeutics.

The intestinal epithelium consists of cells derived from continuously cycling Lgr5hi intestinal stem cells (Lgr5hi ISCs) that mature developmentally in an ordered fashion as the cells progress along the crypt-luminal axis. Perturbed function of Lgr5hi ISCs with aging is documented, but the consequent impact on overall mucosal homeostasis has not been defined. Using single-cell RNA sequencing, the progressive maturation of progeny was dissected in the mouse intestine, which revealed that transcriptional reprogramming with aging in Lgr5hi ISCs retarded the maturation of cells in their progression along the crypt-luminal axis. Importantly, treatment with metformin or rapamycin at a late stage of mouse lifespan reversed the effects of aging on the function of Lgr5hi ISCs and subsequent maturation of progenitors. The effects of metformin and rapamycin overlapped in reversing changes of transcriptional profiles but were also complementary, with metformin more efficient than rapamycin in correcting the developmental trajectory. Therefore, our data identify novel effects of aging on stem cells and the maturation of their daughter cells contributing to the decline of epithelial regeneration and the correction by geroprotectors.

Paneth cells as the origin of intestinal cancer in the context of inflammation.

Paneth cells (PCs), responsible for the secretion of antimicrobial peptides in the small intestine and for niche support to Lgr5+ crypt-base columnar stem cells (CBCs), have been shown to respond to inflammation by dedifferentiating into stem-like cells in order to sustain a regenerative response1,2. Therefore, PCs may represent the cells-of-origin of intestinal cancer in the context of inflammation. To test this hypothesis, we targeted Apc, Kras, and Tp53 mutations in Paneth cells by Cre-Lox technology and modelled inflammation by dextran sodium sulfate (DSS) administration. PC-specific loss of Apc resulted in multiple small intestinal tumors, whereas Kras or Tp53 mutations did not. Compound Apc and Kras mutations in PCs resulted in a striking increase in tumor multiplicity even in the absence of the inflammatory insult. By combining scRNAseq with lineage tracing to capture the conversion of PCs into bona fide tumor cells, we show that they progress through a "revival stem cell" (RSC) state characterized by high Clusterin (Clu) expression and Yap1 signaling, reminiscent of what has been previously observed upon irradiation of the mouse digestive tract3. Accordingly, comparison of PC- and Lgr5-derived murine intestinal tumors revealed differences related to Wnt signaling and inflammatory pathways which match the dichotomy of CBCs and injury-induced RSCs4 between human sporadic colon cancers and those arising in the context of inflammatory bowel diseases. Last, we show that western-style dietary habits, known to trigger a low-grade inflammation throughout the intestinal tract, underlie the analogous dedifferentiation of Paneth cells and their acquisition of stem-like features. Taken together, our results show that intestinal cancer arises in the context of inflammation through the dedifferentiation of committed secretory lineages such as Paneth cells and the activation of the revival stem cell state. As such, a true quiescent stem cell identity may be hidden in fully committed and postmitotic lineages which, upon inflammation, support the regenerative response by re-entering the cell cycle and dedifferentiating into RSCs. The chronic nature of the tissue insult in inflammatory bowel diseases and even in the context of western-style dietary habits is likely to result in the expansion of cell targets for tumor initiation and progression.

The origin of intestinal cancer in the context of inflammation.

According to conventional views, colon cancer originates from stem cells. However, inflammation, a key risk factor for colon cancer, was shown to suppress intestinal stemness. Here, we employed Paneth cells (PCs) as a model to assess the capacity of differentiated lineages to trigger tumorigenesis in the context of inflammation. Upon inflammation, PC-specific Apc mutations led to intestinal tumors reminiscent not only of those arising in inflammatory bowel disease (IBD) patients but also of a larger fraction of sporadic colon cancers. The latter is likely due to the inflammatory consequences of Western-style dietary habits, the major colon cancer risk factor. Computational methods designed to predict the cell-of-origin of cancer confirmed that, in a substantial fraction of sporadic colon cancers the cells-of-origin are secretory lineages and not stem cells.

Dietary cholecalciferol and calcium levels in a Western-style defined rodent diet alter energy metabolism and inflammatory responses in mice.

Male and female C57Bl6 mice were fed a control AIN76A diet, a new Western-style diet (NWD1) reflecting dietary patterns linked to elevated colon cancer incidence (higher fat, lower cholecalciferol, calcium, methyl donors, fiber), or NWD1 with elevated cholecalciferol and calcium (NWD2) from weaning. After 24 wk, serum 25-hydroxyvitamin D [25(OH)D] decreased by >80% in the NWD1 group compared with controls, but with no alteration in serum calcium or bone mineral density. The decreased serum 25(OH)D was prevented in the NWD2 group. After 32 wk, the NWD1 group compared with controls reduced overall energy expenditure by 15% without altering food consumption or physical activity and induced glucose intolerance, phenotypes associated with metabolic syndrome. These responses were unexpectedly exacerbated in the NWD2 group, further shifting mice toward greater fatty acid storage rather than oxidation compared with both control and NWD1 groups, but there was no change in physical activity, causing significant weight gain due to increased fat mass. The NWD1 group also exhibited inflammatory responses compared with controls, including macrophage-associated crown-like structures in epididymal adipose tissue and increased serum concentrations of the proinflammatory cytokine IL-1ß, and of its targets, MCP-1 and Rantes, which were prevented or greatly mitigated in the NWD2 group. However, there was also elevated lipid storage in the liver and steatosis not seen in the control and NWD1 groups. Thus, elevating cholecalciferol and calcium in a Western-style diet can reduce inflammation associated with risk for colon tumor development, but interaction of nutrients in this diet can compromise liver function when fed long term.

MYBL2, a link between proliferation and differentiation in maturing colon epithelial cells.

Multiple signals, controlling both proliferation and differentiation, must be integrated in the reprogramming of intestinal epithelial cells during maturation along the crypt-luminal axis. The v-myb family member Mybl2, a molecule implicated in the development and maintenance of the stem cell phenotype, has been suggested to play an important role in proliferation and differentiation of several cell types and is a gene we have found is commonly regulated in several systems of colon cell maturation both in vitro and in vivo. Here we show that siRNA silencing of Mybl2 in proliferating Caco-2 cells increases expression of the cell-cycle regulators cdk2, cyclin D2, and c-myc and decreases expression of cdc25B and cyclin B2 with a consequent 10% increase of cells in G2/M and a complementary 10% decrease in G1. Mybl2 occupies sequences upstream of transcriptional start sites of cyclin D2, c-myc, cyclin B2, and cdc25B and regulates reporter activity driven by upstream regions of cdk2, cyclin D2, and c-myc. These data suggest that Mybl2 plays a subtle but key role in linking specific aspects of cell-cycle progression with generation of signals for differentiation and may therefore be fundamental in commitment of intestinal epithelial cells to differentiation pathways during their maturation.

Mybl2, downregulated during colon epithelial cell maturation, is suppressed by miR-365.

Altered profiles of gene expression reflect the reprogramming of intestinal epithelial cells during their maturation along the crypt-luminal axis. To focus on genes important in this process, and how they in turn are regulated, we identified 14 transcripts commonly downregulated in expression during lineage-specific maturation of the immortalized cell lines Caco-2 (absorptive), HT29Cl16E (goblet), and HT29Cl19A (secretory) induced by contact inhibition of growth or the short-chain fatty acid butyrate. One such gene, Mybl2 (Myb-related protein B), has been linked to the stem cell phenotype, and we report is also markedly suppressed in maturing cells along the crypt-luminal axis in vivo. Mybl2 is not significantly downregulated transcriptionally during colon cell maturation, but we identified a potential micro-RNA (miRNA)-binding sequence in the Mybl2 3'-untranslated region that mediates reporter gene suppression in differentiating colon cells. Accordingly, miRNAs predicted to bind this functional target are upregulated in differentiating colon epithelial cells in vitro and in vivo; expression of one of these, hsa-miR-365 (but not hsa-324-5p), suppresses Mybl2 protein expression in proliferating Caco-2 cells. These data demonstrate that miRNA silencing plays an important role in regulating gene expression in maturing colon epithelial cells, and that utilizing a target-centered approach, rather than profiling global miRNA expression, can identify physiologically relevant, functional miRNAs.

Intestinal epithelial-specific PTEN inactivation results in tumor formation.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of phosphatidylinositol 3-kinase (PI3K) signaling that is frequently inactivated in colorectal cancer through mutation, loss of heterozygosity, or epigenetic mechanisms. The aim of this study was to determine the effect of intestinal-specific PTEN inactivation on intestinal epithelial homeostasis and tumorigenesis. PTEN was deleted specifically in the intestinal epithelium, by crossing PTEN(Lox/Lox) mice with villin(Cre) mice. PTEN was robustly expressed in the intestinal epithelium and maximally in the differentiated cell compartment. Targeted inactivation of PTEN in the intestinal epithelium of PTEN(Lox/Lox)/villin(Cre) mice was confirmed by genotyping, immunohistochemistry, and qPCR. While intestinal-specific PTEN deletion did not have a major effect on cell fate determination or proliferation in the small intestine, it did increase phosphorylated (p) protein kinase B (AKT) expression in the intestinal epithelium, and 19% of animals developed small intestinal adenomas and adenocarcinomas at 12 mo of age. These tumors demonstrated pAKT and nuclear ß-catenin staining, indicating simultaneous activation of the PI3K/AKT and Wnt signaling pathways. These findings demonstrate that, while PTEN inactivation alone has a minimal effect on intestinal homeostasis, it can facilitate tumor promotion upon deregulation of ß-catenin/TCF signaling, further establishing PTEN as a bona fide tumor suppressor gene in intestinal cancer.

Transition state analogue of MTAP extends lifespan of APCMin/+ mice.

A mouse model of human Familial Adenomatous Polyposis responds favorably to pharmacological inhibition of 5'-methylthioadenosine phosphorylase (MTAP). Methylthio-DADMe-Immucillin-A (MTDIA) is an orally available, transition state analogue inhibitor of MTAP. 5'-Methylthioadenosine (MTA), the substrate for MTAP, is formed in polyamine synthesis and is recycled by MTAP to S-adenosyl-L-methionine (SAM) via salvage pathways. MTDIA treatment causes accumulation of MTA, which inhibits growth of human head and neck (FaDu) and lung (H359, A549) cancers in immunocompromised mouse models. We investigated the efficacy of oral MTDIA as an anti-cancer therapeutic for intestinal adenomas in immunocompetent APCMin/+ mice, a murine model of human Familial Adenomatous Polyposis. Tumors in APCMin/+ mice were decreased in size by MTDIA treatment, resulting in markedly improved anemia and doubling of mouse lifespan. Metabolomic analysis of treated mice showed no changes in polyamine, methionine, SAM or ATP levels when compared with control mice but indicated an increase in MTA, the MTAP substrate. Generation of an MTDIA-resistant cell line in culture showed a four-fold amplification of the methionine adenosyl transferase (MAT2A) locus and expression of this enzyme. MAT2A is downstream of MTAP action and catalyzes synthesis of the SAM necessary for methylation reactions. Immunohistochemical analysis of treated mouse intestinal tissue demonstrated a decrease in symmetric dimethylarginine, a PRMT5-catalyzed modification. The anti-cancer effects of MTDIA indicate that increased cellular MTA inhibits PRMT5-mediated methylations resulting in attenuated tumor growth. Oral dosing of MTDIA as monotherapy has potential for delaying the onset and progression of colorectal cancers in Familial Adenomatous Polyposis (FAP) as well as residual duodenal tumors in FAP patients following colectomy. MTDIA causes a physiologic inactivation of MTAP and may also have efficacy in combination with inhibitors of MAT2A or PRMT5, known synthetic-lethal interactions in MTAP-/- cancer cell lines.

Gamma delta T cells provide an early source of interferon gamma in tumor immunity.

Interferon (IFN)-gamma is necessary for tumor immunity, however, its initial cellular source is unknown. Because gammadelta T cells primarily produce this cytokine upon activation, we hypothesized that they would provide an important early source of IFN-gamma in tumor immunosurveillance. To address this hypothesis, we first demonstrated that gammadelta T cell-deficient mice had a significantly higher incidence of tumor development after challenge with a chemical carcinogen methylcholanthrene (MCA) or inoculation with the melanoma cell line B16. In wild-type mice, gammadelta T cells were recruited to the site of tumor as early as day 3 after inoculation, followed by alphabeta T cells at day 5. We then used bone marrow chimeras and fetal liver reconstitutions to create mice with an intact gammadelta T cell repertoire but one that was specifically deficient in the capacity to produce IFN-gamma. Such mice had a higher incidence of tumor development, induced either with MCA or by inoculation of B16 melanoma cells, compared with mice with IFN-gamma-competent gammadelta T cells. Moreover, genetic deficiency of gammadelta T cells resulted in impaired IFN-gamma production by tumor antigen-triggered alphabeta T cell upon immunization with tumor lysate. These results demonstrate that gammadelta T cells can play a necessary role in tumor immunity through provision of an early source of IFN-gamma that in turn may regulate the function of tumor-triggered alphabeta T cells.

PKCalpha tumor suppression in the intestine is associated with transcriptional and translational inhibition of cyclin D1.

Alterations in PKC isozyme expression and aberrant induction of cyclin D1 are early events in intestinal tumorigenesis. Previous studies have identified cyclin D1 as a major target in the antiproliferative effects of PKCalpha in non-transformed intestinal cells; however, a link between PKC signaling and cyclin D1 in colon cancer remained to be established. The current study further characterized PKC isozyme expression in intestinal neoplasms and explored the consequences of restoring PKCalpha or PKCdelta in a panel of colon carcinoma cell lines. Consistent with patterns of PKC expression in primary tumors, PKCalpha and delta levels were generally reduced in colon carcinoma cell lines, PKCbetaII was elevated and PKCepsilon showed variable expression, thus establishing the suitability of these models for analysis of PKC signaling. While colon cancer cells were insensitive to the effects of PKC agonists on cyclin D1 levels, restoration of PKCalpha downregulated cyclin D1 by two independent mechanisms. PKCalpha expression consistently (a) reduced steady-state levels of cyclin D1 by a novel transcriptional mechanism not previously seen in non-transformed cells, and (b) re-established the ability of PKC agonists to activate the translational repressor 4E-BP1 and inhibit cyclin D1 translation. In contrast, PKCdelta had modest and variable effects on cyclin D1 steady-state levels and failed to restore responsiveness to PKC agonists. Notably, PKCalpha expression blocked anchorage-independent growth in colon cancer cells via a mechanism partially dependent on cyclin D1 deficiency, while PKCdelta had only minor effects. Loss of PKCalpha and effects of its re-expression were independent of the status of the APC/beta-catenin signaling pathway or known genetic alterations, indicating that they are a general characteristic of colon tumors. Thus, PKCalpha is a potent negative regulator of cyclin D1 expression and anchorage-independent cell growth in colon tumor cells, findings that offer important perspectives on the frequent loss of this isozyme during intestinal carcinogenesis.