Marshall Behrens

Immune-induced epithelial to mesenchymal transition in vivo generates breast cancer stem cells

The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell-dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24(-/lo)CD44(+) phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have several important implications related to disease progression and relapse.

TGFβ/TNFα-Mediated Epithelial–Mesenchymal Transition Generates Breast Cancer Stem Cells with a Claudin-Low Phenotype

Breast cancer recurrence is believed to be caused by a subpopulation of cancer cells that possess the stem cell attribute of treatment resistance. Recently, we and others have reported the generation of breast cancer stem cells (BCSC) by epithelial-mesenchymal transition (EMT), although the physiologic process by which these cells may arise in vivo remains unclear. We show here that exposure of tumor cells to TGFβ and TNFα induces EMT and, more importantly, generates cells with a stable BCSC phenotype which is shown by increased self-renewing capacity, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide, and paclitaxel. Furthermore, gene expression analyses found that the TGFβ/TNFα-derived BCSCs showed downregulated expression of genes encoding claudin 3, 4, and 7 and the luminal marker, cytokeratin 18. These changes indicate a shift to the claudin-low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlated with a poor prognosis. Taken together, the data show that cytokine exposure can be used to generate stable BCSCs ex vivo, and suggest that these cells may provide a valuable tool in the identification of stem cell-directed biomarkers and therapies in breast cancer.

Tumor-Infiltrating Programmed Death Receptor-1+ Dendritic Cells Mediate Immune Suppression in Ovarian Cancer

Within the ovarian cancer microenvironment, there are several mechanisms that suppress the actions of antitumor immune effectors. Delineating the complex immune microenvironment is an important goal toward developing effective immune-based therapies. A dominant pathway of immune suppression in ovarian cancer involves tumor-associated and dendritic cell (DC)-associated B7-H1. The interaction of B7-H1 with PD-1 on tumor-infiltrating T cells is a widely cited theory of immune suppression involving B7-H1 in ovarian cancer. Recent studies suggest that the B7-H1 ligand, programmed death receptor-1 (PD-1), is also expressed on myeloid cells, complicating interpretations of how B7-H1 regulates DC function in the tumor. In this study, we found that ovarian cancer-infiltrating DCs progressively expressed increased levels of PD-1 over time in addition to B7-H1. These dual-positive PD-1+ B7-H1+ DCs have a classical DC phenotype (i.e., CD11c+CD11b+CD8−), but are immature, suppressive, and respond poorly to danger signals. Accumulation of PD-1+B7-H1+ DCs in the tumor was associated with suppression of T cell activity and decreased infiltrating T cells in advancing tumors. T cell suppressor function of these DCs appeared to be mediated by T cell-associated PD-1. In contrast, ligation of PD-1 expressed on the tumor-associated DCs suppressed NF-κB activation, release of immune regulatory cytokines, and upregulation of costimulatory molecules. PD-1 blockade in mice bearing ovarian cancer substantially reduced tumor burden and increased effector Ag-specific T cell responses. Our results reveal a novel role of tumor infiltrating PD-1+B7-H1+ DCs in mediating immune suppression in ovarian cancer.

AXL induces epithelial-to-mesenchymal transition and regulates the function of breast cancer stem cells

Despite significant progress in the treatment of breast cancer, particularly through the use of targeted therapy, relapse and chemoresistance remain a major hindrance to the fight to minimize the burden of the disease. It is becoming increasingly clear that a rare subpopulation of cells known as cancer stem cells (CSC), able to be generated through epithelial-to-mesenchymal transition (EMT) and capable of tumor initiation and self-renewal, contributes to treatment resistance and metastases. This means that a more effective therapy should target both the chemoresistant CSCs and the proliferating epithelial cells that give rise to them to reverse EMT and to attenuate their conversion to CSCs. Here, we demonstrate a novel function of AXL in acting upstream to induce EMT in normal and immortalized human mammary epithelial cells in an apparent positive feedback loop mechanism and regulate breast CSC (BCSC) self-renewal and chemoresistance. Downregulation of AXL using MP470 (Amuvatinib) reversed EMT in mesenchymal normal human mammary epithelial cells and murine BCSCs attenuating self-renewal and restored chemosensitivity of the BCSCs. AXL expression was also found to be associated with the expression of stem cell genes, regulation of metastases genes, increased tumorigenicity and was important for BCSC invasion and migration. Inactivation of AXL also led to the downregulation of nuclear factor-κB pathway and reduced tumor formation in vivo. Taken together, our data suggest that targeted therapy against AXL, in combination with systemic therapies, has the potential to improve response to anticancer therapies and to reduce breast cancer recurrence and metastases.

Immunoediting of Cancers May Lead to Epithelial to Mesenchymal Transition

Tumors evade both natural and pharmacologically induced (e.g., vaccines) immunity by a variety of mechanisms, including induction of tolerance and immunoediting. Immunoediting results in reshaping the immunogenicity of the tumor, which can be accompanied by loss of Ag expression and MHC molecules. In this study, we evaluated immunoediting in the neu-transgenic mouse model of breast cancer. A tumor cell line that retained expression of rat neu was generated from a spontaneous tumor of the neu-transgenic mouse and, when injected into the non-transgenic parental FVB/N mouse, resulted in the development of a strong immune response, initial rejection, and ultimately the emergence of neu Ag-loss variants. Morphologic and microarray data revealed that the immunoedited tumor cells underwent epithelial to mesenchymal transition accompanied by an up-regulation of invasion factors and increased invasiveness characteristic of mesenchymal tumor cells. These results suggest that immunoediting of tumor results in cellular reprogramming may be accompanied by alterations in tumor characteristics including increased invasive potential. Understanding the mechanisms by which tumors are immunoedited will likely lead to a better understanding of how tumors evade immune detection.

Accumulation of Memory Precursor CD8 T Cells in Regressing Tumors following Combination Therapy with Vaccine and Anti-PD-1 Antibody

Immunosuppression in the tumor microenvironment blunts vaccine-induced immune effectors. PD-1/B7-H1 is an important inhibitory axis in the tumor microenvironment. Our goal in this study was to determine the effect of blocking this inhibitory axis during and following vaccination against breast cancer. We observed that using anti-PD-1 antibody and a multipeptide vaccine (consisting of immunogenic peptides derived from breast cancer antigens, neu, legumain, and β-catenin) as a combination therapy regimen for the treatment of breast cancer-bearing mice prolonged the vaccine-induced progression-free survival period. This prolonged survival was associated with increase in number of Tc1 and Tc2 CD8 T cells with memory precursor phenotype, CD27+IL-7RhiT-betlo, and decrease in number of PD-1+ dendritic cells (DC) in regressing tumors and enhanced antigen reactivity of tumor-infiltrating CD8 T cells. It was also observed that blockade of PD-1 on tumor DCs enhanced IL-7R expression on CD8 T cells. Taken together, our results suggest that PD-1 blockade enhances breast cancer vaccine efficacy by altering both CD8 T cell and DC components of the tumor microenvironment. Given the recent success of anti-PD-1 monotherapy, our results are encouraging for developing combination therapies for the treatment of patients with cancer in which anti-PD-1 monotherapy alone may be ineffective (i.e., PD-L1-negative tumors).

CD4 and CD8 T Cells in Susceptibility/Protection to Collagen-Induced Arthritis in HLA-DQ8-Transgenic Mice: Implications for Rheumatoid Arthritis

To investigate the role of CD4 and CD8 T cells in arthritis, we generated transgenic mice deficient in CD4 and CD8 molecules expressing RA-susceptible gene HLA-DQ8. DQ8·CD4−/− mice were resistant to developing collagen-induced arthritis (CIA). However, DQ8·CD8−/− mice developed CIA with increased incidence and more severity than DQ8 mice. Both DQ8·CD8−/− and DQ8 mice produced rheumatoid factor. In addition, DQ8·CD8−/− mice produced antinuclear Abs. The B cell compartment and expression of DQ8 were normal in all the strains, although frequency of cells expressing DQ8 was less in CD4−/− mice. An increased frequency of CD3+ double-negative (DN) T cells was found in DQ8·CD8−/− compared with DQ8·CD4−/− and DQ8 mice. These CD3+ DN T cells produced high amounts of IL-10 in CD8-deficient mice. Analysis of cell division using a cell cycle tracking dye showed a higher rate of division of CD3+ and CD3+ DN T cells in DQ8·CD8−/− mice compared with DQ8·CD4−/− and DQ8 mice. Decreased apoptosis was seen in CIA-susceptible DQ8 and CD8-deficient mice, indicating a defect in activation-induced cell death. These observations suggest that CD4 cells are necessary for initiation of CIA in DQ8 mice. We hypothesize that CD8+ T cells are not capable of initiating CIA in DQ8-transgenic mice but may have a regulatory/protective effect.

Delineating the Role of the HLA-DR4 “Shared Epitope” in Susceptibility versus Resistance to Develop Arthritis

In humans, HLA-DR alleles sharing amino acids at the third hypervariable region with DRB1*0401(shared epitope) are associated with a predisposition to rheumatoid arthritis, whereas DRB1*0402 is not associated with such a predisposition. Both DRB1*0402 and DRB1*0401 occur in linkage with DQ8 (DQB1*0302). We have previously shown that transgenic (Tg) mice expressing HLA-DRB1*0401 develop collagen-induced arthritis. To delineate the role of “shared epitope” and gene complementation between DR and DQ in arthritis, we generated DRB1*0402, DRB1*0401.DQ8, and DRB1*0402.DQ8 Tg mice lacking endogenous class II molecules, AE°. DRB1*0402 mice are resistant to develop arthritis. In double-Tg mice, the DRB1*0401 gene contributes to the development of collagen-induced arthritis, whereas DRB1*0402 prevents the disease. Humoral response to type II collagen is not defective in resistant mice, although cellular response to type II collagen is lower in *0402 mice compared with *0401 mice. *0402 mice have lower numbers of T cells in thymus compared with *0401 mice, suggesting that the protective effect could be due to deletion of autoreactive T cells. Additionally, DRB1*0402 mice have a higher number of regulatory T cells and show increased activation-induced cell death, which might contribute toward protection. In DRB1*0401.DQ8 mice, activated CD4+ T cells express class II genes and can present DR4- and DQ8-restricted peptides in vitro, suggesting a role of class II+ CD4 T cells locally in the joints. The data suggest that polymorphism in DRB1 genes determines predisposition to develop arthritis by shaping the T cell repertoire in thymus and activating autoreactive or regulatory T cells.

Mechanism by which HLA-DR4 Regulates Sex-bias of Arthritis in humanized mice

HLA class II allele DRB1*0401 is associated with predisposition to Rheumatoid Arthritis in humans as well as collagen-induced arthritis in mice. Predominantly females develop arthritis in humans and DR4 transgenic mice; however the mechanism of sex-bias is still unknown. We have investigated the molecular basis by which DR4 is associated with sex-bias of arthritis. Here we show that differential antigen-specific immune mechanisms in DR4 male and female mice lead to increased susceptibility in female mice. B cells are hyperactive and present DR-restricted peptides robustly in females compared to males. Antigen-specific response showed that females produced B cell modulating cytokines like IL-13 while males produced IFNgamma. Male transgenic mice have higher number of T and B regulatory cells. An exogenous supply of 17beta estradiol in male mice led to enhanced expression of DR4 and antigen-specific response to DR4-restricted peptides. On the other hand, castration increased the incidence of arthritis. We propose that sex-bias in arthritis involves B cells and presentation of antigen by HLA-DR4 leading to activation of autoreactive cells and autoantibodies production in females, while regulatory B cells in males protect them from pathogenesis. The transgenic mice expressing RA susceptible haplotype simulate human RA and may be valuable to study gender differences observed in patients.

Cellular and humoral immunity in arthritis are profoundly influenced by the interaction between cigarette smoke effects and host HLA-DR and DQ genes

Individuals carrying DRB1*0401 who smoke cigarettes are at an increased risk of developing severe seropositive RA. To determine how cigarette smoke (CS) interacts with host genetic factors in the induction of RA-associated autoimmunity, we used transgenic mice carrying the RA-susceptible HLA genes DR4 and DQ8, but lacking all endogenous murine class II molecules. Cigarette smoke exposure augmented peptidylarginine deiminase (PAD) enzyme expression, and enhanced immune responses to citrullinated collagen and vimentin. Here we show for the first time that DQ molecules can present citrullinated peptides much more efficiently than native peptides. Interestingly, CS exposure suppressed collagen-induced arthritis (CIA) in DRB1*0401 mice although innate immune response was enhanced. On the other hand, CS exposure exacerbated CIA in DQ8 mice, which was accompanied by an increased expression of Th17 gene transcripts in lungs. These observations suggest that cigarette smoke promotes antigen-specific autoimmunity that is profoundly influenced by host genetic factors.