Varada P. Rao

Mast cells are an essential hematopoietic component for polyp development

It is generally agreed that most colon cancers develop from adenomatous polyps, and it is this fact on which screening strategies are based. Although there is overwhelming evidence to link intrinsic genetic lesions with the formation of these preneoplastic lesions, recent data suggest that the tumor stromal environment also plays an essential role in this disease. In particular, it has been suggested that CD34+ immature myeloid precursor cells are required for tumor development and invasion. Here we have used mice conditional for the stabilization of β-catenin or defective for the adenomatous polyposis coli (APC) gene to reinvestigated the identity and importance of tumor-infiltrating hematopoietic cells in polyposis. We show that, from the onset, polyps are infiltrated with proinflammatory mast cells (MC) and their precursors. Depletion of MC either pharmacologically or through the generation of chimeric mice with genetic lesions in MC development leads to a profound remission of existing polyps. Our data suggest that MC are an essential hematopoietic component for preneoplastic polyp development and are a novel target for therapeutic intervention.

CD4+CD25+ Regulatory Lymphocytes Induce Regression of Intestinal Tumors in ApcMin/+ Mice

Colorectal cancer in humans results from sequential genetic changes in intestinal epithelia commencing with inactivation of the APC tumor suppressor gene. Roles for host immunity in epithelial tumorigenesis are poorly understood. It has been previously shown that CD4+CD25+ lymphocytes inhibit colitis-associated epithelial tumors in Rag-deficient mice. Here we show that addition of CD4+CD25+ lymphocytes in ApcMin/+ mice reduces multiplicity of epithelial adenomas. Interleukin-10 was required in regulatory cells for therapeutic effect. Recipients of regulatory cells showed increased apoptosis and down-regulation of cyclooxygenase-2 within tumors coincident with tumor regression. These data suggest a role for regulatory lymphocytes in epithelial homeostasis in the ApcMin/+ mouse model of intestinal polyposis. Similarities with cancer of the breast, prostate, lung, and other sites raise the possibility of broader roles for regulatory lymphocytes in prevention and treatment of epithelial cancers in humans.

Nitric oxide and TNF-alpha trigger colonic inflammation and carcinogenesis in Helicobacter hepaticus-infected, Rag2-deficient mice.

Recombinase-activating gene-2-deficient (Rag2−/−) mice lacking functional lymphocytes provide a useful model of chronic inflammatory bowel disease-emulating events in human colon cancer. Infection of Rag2−/− mice with Helicobacter hepaticus led to accumulation of macrophages and neutrophils in the colon, a process temporally related to up-regulation of tissue inducible nitric oxide synthase (iNOS) expression at the site of infection and increased nitric oxide (NO) production, as evidenced by urinary excretion of nitrate. Progressive development of increasingly severe inflammation, hyperplasia, dysplasia, and cancer accompanied these changes. Concurrent administration of an iNOS inhibitor prevented NO production and abrogated epithelial pathology and inhibited the onset of cancer. The presence of Gr-1+ neutrophils and elevated tumor necrosis factor-α (TNF-α) expression in colon were required for increased iNOS expression and cancer, whereas interleukin-10 (IL-10) down-regulated TNF-α and iNOS expression and suppressed cancer. Anti-inflammatory CD4+ regulatory lymphocytes also down-regulated iNOS and reduced cancer formation. Collectively, these results confirm essential roles for inflammation, increased TNF-α expression, and elevated NO production in colon carcinogenesis.

Innate Immune Inflammatory Response against Enteric Bacteria Helicobacter hepaticus Induces Mammary Adenocarcinoma in Mice

Inflammation associated with bacterial infections is a risk factor for cancers in humans, yet its role in breast cancer remains poorly understood. We have previously shown that innate immune inflammatory response against intestinal bacteria is sufficient to induce colon cancer. Here we report that infecting Rag2-deficient C57BL/6 Apc(Min/+) mice with an intestinal bacterial pathogen, Helicobacter hepaticus, significantly promotes mammary carcinoma in females and enhances intestinal adenoma multiplicity by a tumor necrosis factor alpha (TNFalpha)-dependent mechanism. The mammary and intestinal tumor development as well as the increase in proinflammatory mediators is suppressed by adoptive transfer of interleukin 10-competent CD4+CD45RB(lo)CD25+ regulatory (T(R)) cells. Furthermore, prior exposure of donor mice to H. hepaticus significantly enhances antitumor potency of their T(R) cells. Interestingly, these microbially experienced T(R) cells suppress tumorigenesis more effectively in recipient mice irrespective of their tumor etiology. These data suggest that infections with enteric pathogens enhance T(R)-cell potency and protect against epithelial cancers later in life, potentially explaining paradoxical increases in cancer risk in developed countries having more stringent hygiene practices. The possibility that dysregulated gut microbial infections in humans may lead to cancer in anatomically distant organs, such as breast, highlights the need for novel immune-based strategies in cancer prevention and treatment.

Unifying roles for regulatory T cells and inflammation in cancer

Activities of CD4+ regulatory (TREG) cells restore immune homeostasis during chronic inflammatory disorders. Roles for TREG cells in inflammation‐associated cancers, however, are paradoxical. It is widely believed that TREG function in cancer mainly to suppress protective anticancer responses. However, we demonstrate here that TREG cells also function to reduce cancer risk throughout the body by efficiently downregulating inflammation arising from the gastrointestinal (GI) tract. Building on a “hygiene hypothesis” model in which GI infections lead to changes in TREG that reduce immune‐mediated diseases, here we show that gut bacteria‐triggered TREG may function to inhibit cancer even in extraintestinal sites. Ability of bacteria‐stimulated TREG to suppress cancer depends on interleukin (IL)‐10, which serves to maintain immune homeostasis within bowel and support a protective antiinflammatory TREG phenotype. However, under proinflammatory conditions, TREG may fail to provide antiinflammatory protection and instead contribute to a T helper (Th)‐17‐driven procarcinogenic process; a cancer state that is reversible by downregulation of inflammation. Consequently, hygienic individuals with a weakened IL‐10 and TREG‐mediated inhibitory loop are highly susceptible to the carcinogenic consequences of elevated IL‐6 and IL‐17 and show more frequent inflammation‐associated cancers. Taken together, these data unify seemingly divergent disease processes such as autoimmunity and cancer and help explain the paradox of TREG and inflammation in cancer. Enhancing protective TREG functions may promote healthful longevity and significantly reduce risk of cancer.

Proinflammatory CD4+CD45RBhi Lymphocytes Promote Mammary and Intestinal Carcinogenesis in ApcMin/+ Mice

Cancers of breast and bowel are increasingly frequent in humans. Chronic inflammation is known to be a risk factor for these malignancies, yet cellular and molecular mechanisms linking inflammation and carcinogenesis remain poorly understood. Here, we apply a widely used T-cell transfer paradigm, involving adoptive transfer of proinflammatory CD4+CD45RBhi (TE) cells to induce inflammatory bowel disease (IBD) in mice, to investigate roles of inflammation on carcinogenesis in the ApcMin/+ mouse model of intestinal polyposis. We find that transfer of TE cells significantly increases adenoma multiplicity and features of malignancy in recipient ApcMin/+ mice. Surprisingly, we find that female ApcMin/+ recipients of TE cells also rapidly develop mammary tumors. Both intestinal polyposis and mammary adenocarcinoma are abolished by cotransfer of anti-inflammatory CD4+CD45RBlo regulatory lymphocytes or by neutralization of key proinflammatory cytokine tumor necrosis factor-α. Lastly, down-regulation of cyclooxygenase-2 and c-Myc expression is observed coincident with tumor regression. These findings define a novel mouse model of inflammation-driven mammary carcinoma and suggest that epithelial carcinogenesis can be mitigated by anti-inflammatory cells and cytokines known to regulate IBD in humans and mice. (Cancer Res 2006; 66(1): 57-61)

Breast Cancer: Should Gastrointestinal Bacteria Be on Our Radar Screen?

Anti-inflammatory drugs and antibiotics alter the risk of breast cancer in women, but roles for bacteria and inflammation in breast malignancies are poorly understood. A recent study in mice suggests that intestinal bacteria can trigger mammary carcinoma. The mechanisms involved in this effect suggest that dysregulated host immune responses to enteric bacteria can influence the development of extraintestinal cancers, highlighting the opportunities for prevention and treatment aimed at promoting intestinal homeostasis.

Wild-Type and Interleukin-10-Deficient Regulatory T Cells Reduce Effector T-Cell-Mediated Gastroduodenitis in Rag2−/− Mice, but Only Wild-Type Regulatory T Cells Suppress Helicobacter pylori Gastritis

ABSTRACT CD4+ CD45RBhi CD25− effector T cells (TE) promote Helicobacter pylori gastritis in mice, and CD4+ CD45RBlo CD25+ regulatory T cells (TR) are anti-inflammatory. Using adoptive transfer into H. pylori-infected Rag2−/− mice, we evaluated effects of wild-type (wt) C57BL/6 or congenic interleukin-10-deficient (IL-10−/−) TR cells on gastritis, gastric cytokines, and H. pylori colonization. Infected Rag2−/− mice colonized in the corpus and antrum with 105 to 106H. pylori CFU/gram without associated gastritis. TE cell transfer caused morbidity and an H. pylori-independent pangastritis and duodenitis (gastroduodenitis) associated with increased expression of gamma interferon (IFN-γ) and tumor necrosis factor alpha. TE cell transfer to H. pylori-infected mice led to additive corpus gastritis associated with inflammatory cytokine expression and reduced colonization. wt TR cells reduced morbidity, H. pylori corpus gastritis, gastroduodenitis, and inflammatory cytokine expression and reversed the decline in H. pylori colonization attributable to TE cells. Although less effective than wt TR cells, IL-10−/− TR cells also reduced morbidity and gastroduodenitis but did not reduce H. pylori corpus gastritis or impact TE cell inhibition of colonization. Gastric tissues from mice receiving wt TR cells expressed higher levels of Foxp3 compared to recipients of IL-10−/− TR cells, consistent with lower regulatory activity of IL-10−/− TR cells. These results demonstrate that wt TR cells suppressed TE-cell-mediated H. pylori-independent gastroduodenitis and H. pylori-dependent corpus gastritis more effectively than IL-10−/− TR cells. Compartmental differences in TE-cell- and H. pylori-mediated inflammation and in regulatory effects between wt TR and IL-10−/− TR cells suggest that IL-10 expression by wt TR cells is important to regulatory suppression of gastric inflammation.

Mutations in bone marrow-derived stromal stem cells unmask latent malignancy

Neoplastic epithelia may remain dormant and clinically unapparent in human patients for decades. Multiple risk factors including mutations in tumor cells or the stromal cells may affect the switch from dormancy to malignancy. Gene mutations, including p53 mutations, within the stroma of tumors are associated with a worse clinical prognosis; however, it is not known if these stromal mutations can promote tumors in genetically at-risk tissue. To address this question, Apc(Min/+) and Apc(Min/+) Rag2(-/-) mice, which have a predilection to mammary carcinoma (as well as wild-type (wt) mice), received mesenchymal stem cells (MSC) with mutant p53 (p53MSC) transferred via tail vein injection. In the wt mouse, p53MSC circulated in the periphery and homed to the marrow cavity where they could be recovered up to a year later without apparent effect on the health of the mouse. No mammary tumors were found. However, in mice carrying the Apc(Min/+) mutation, p53MSC homed to mammary tissue and significantly increased the incidence of mammary carcinoma. Tumor necrosis factor (TNF)-alpha-dependent factors elaborated from mesenchymal cells converted quiescent epithelia into clinically apparent disease. The increased cancer phenotype was completely preventable with neutralization of TNF-alpha or by transfer of CD4(+) regulatory T cells from immune competent donors, demonstrating that immune competency to regulate inflammation was sufficient to maintain neoplastic dormancy even in the presence of oncogenic epithelial and stromal mutations. The significant synergy between host immunity and mesenchymal cells identified here may restructure treatments to restore an anticancer microenvironment.

CD4+ lymphocytes modulate prostate cancer progression in mice

Chronic inflammation contributes to the development of prostate cancer in humans. Here, we show that male ApcMin/+ mice also develop prostate carcinoma with increasing age, mimicking that seen in humans in their 5th or 6th decade of life. Proinflammatory cytokines were significantly linked with cancer and increasing age in our mouse model; however, prostate and bowel tissues lacked evidence of inflammatory cell infiltrates other than mast cells. Lymphocytes protected against cancer, and protection from prostate cancer resided in antiinflammatory CD4+CD25+ regulatory (TREG) cells that downregulated inflammatory cytokines. Supplementation with syngeneic TREG cells collected from wild‐type mice reduced the levels of interleukin (IL)‐6 (p < 0.05) and IL‐9 (p < 0.001) and lowered prostate cancer risk (p < 0.05). Depletion of CD25+ cells in 2‐month‐old animals increased the expression of IL‐6 (p < 0.005) within prostate and increased the frequency of high‐grade prostatic intraepithelial neoplasia (p < 0.05) and microinvasive prostatic carcinoma (p < 0.05) in dorsolateral prostate. Depletion of CD25+ cells in young animals also increased the frequency of intestinal cancer in Min mice. Taken together, chronically elevated proinflammatory cytokines promoted carcinoma in ApcMin/+ mice. TREG lymphocytes downregulated inflammation‐associated carcinogenic processes and contributed to immune and epithelial homeostasis.