A WNT4- and DKK3-driven canonical to noncanonical Wnt signaling switch controls multiciliogenesis.

Multiciliated cells contain hundreds of cilia whose directional movement powers the mucociliary clearance of the airways, a vital host defense mechanism. Multiciliated cell specification requires canonical Wnt signaling, which then must be turned off. Next, ciliogenesis and polarized ciliary orientation are regulated by noncanonical Wnt/planar cell polarity (Wnt/PCP) signaling. The mechanistic relationship between the Wnt pathways is unknown. We show that DKK3, a secreted canonical Wnt regulator and WNT4, a noncanonical Wnt ligand act together to facilitate a canonical to noncanonical Wnt signaling switch during multiciliated cell formation. In primary human airway epithelial cells, DKK3 and WNT4 CRISPR knockout blocks, whereas ectopic expression promotes, multiciliated cell formation by inhibiting canonical Wnt signaling. Wnt4 and Dkk3 single-knockout mice also display defective ciliated cells. DKK3 and WNT4 are co-secreted from basal stem cells and act directly on multiciliated cells via KREMEN1 and FZD6, respectively. We provide a novel mechanism that links specification to cilium biogenesis and polarization for proper multiciliated cell formation.

Inhibition of the hedgehog pathway targets the tumor-associated stroma in pancreatic cancer.

PURPOSE: The Hedgehog (Hh) pathway has emerged as an important pathway in multiple tumor types and is thought to be dependent on a paracrine signaling mechanism. The purpose of this study was to determine the role of pancreatic cancer-associated fibroblasts (human pancreatic stellate cells, HPSCs) in Hh signaling. In addition, we evaluated the efficacy of a novel Hh antagonist, AZD8542, on tumor progression with an emphasis on the role of the stroma compartment. EXPERIMENTAL DESIGN: Expression of Hh pathway members and activation of the Hh pathway were analyzed in both HPSCs and pancreatic cancer cells. We tested the effects of Smoothened (SMO) inhibition with AZD8542 on tumor growth in vivo using an orthotopic model of pancreatic cancer containing varying amounts of stroma. RESULTS: HPSCs expressed high levels of SMO receptor and low levels of Hh ligands, whereas cancer cells showed the converse expression pattern. HPSC proliferation was stimulated by Sonic Hedgehog with upregulation of downstream GLI1 mRNA. These effects were abrogated by AZD8542 treatment. In an orthotopic model of pancreatic cancer, AZD8542 inhibited tumor growth only when HPSCs were present, implicating a paracrine signaling mechanism dependent on stroma. Further evidence of paracrine signaling of the Hh pathway in prostate and colon cancer models is provided, demonstrating the broader applicability of our findings. CONCLUSION: Based on the use of our novel human-derived pancreatic cancer stellate cells, our results suggest that Hh-targeted therapies primarily affect the tumor-associated stroma, rather than the epithelial compartment.

Depletion of CD4+CD25+ regulatory T cells promotes a tumor-specific immune response in pancreas cancer-bearing mice.

BACKGROUND: Pancreas cancer-bearing mice have an increased prevalence of immunosuppressive CD4(+)CD25(+) regulatory T cells (T(reg)). Depletion of T(reg) results in smaller tumors and prolonged host survival. The objective of this study was to evaluate the tumor-specific immune response after depletion of T(reg) alone or in combination with a cancer vaccine. METHODS: Four groups of C57BL/6 mice were challenged with pancreas adenocarcinoma cells (Pan02). The mice received four combinations of antibody-mediated T(reg) depletion and whole tumor cell vaccination: (1) no treatment, (2) T(reg) depletion only, (3) vaccination only, or (4) T(reg) depletion and vaccination. Splenocytes and lymphocytes from tumor-draining lymph nodes were analyzed for tumor-specific release of interferon gamma by enzyme-linked immunosorbent spot assay. RESULTS: In T(reg)-depleted and vaccinated mice, a strong statistical trend toward smaller tumors (P = .05) and longer survival (P = .054) was found compared with untreated mice. T(reg)-depleted mice showed significantly more tumor-specific cells than undepleted mice (P = .02). The number of tumor-specific cells was significantly higher in tumor-draining lymph nodes than in the spleen (P = .002). Similarly, significantly more tumor-specific cells were found in spleens of T(reg)-depleted and vaccinated mice than in vaccinated-only mice (P = .009). CONCLUSIONS: Depletion of T(reg) alone or in combination with a whole tumor cell vaccine promotes a tumor-specific immune response. Thus, strategies incorporating T(reg) depletion might improve the efficacy of cancer vaccines.

Increased prevalence of regulatory T cells (Treg) is induced by pancreas adenocarcinoma.

We reported earlier that patients with breast or pancreas cancer have an increased prevalence of regulatory T cells (Treg) in the blood and tumor draining lymph nodes (TDLNs) compared with healthy individuals. In the current study, we tested the hypothesis that tumor cells promote the prevalence of Treg. The transforming growth factor-beta (TGF-beta) secreting murine pancreas adenocarcinoma, Pan02 cell line was injected into syngeneic C57BL/6 mice and the prevalence of Treg in the TDLNs and tumor spleen was measured weekly. Compared with control mice, the prevalence of CD25+ CD4+ cells in TDLNs and in tumor spleen increased with tumor growth. Analysis of these CD25+ CD4+ T cells in vitro confirmed expression of the Treg marker, Foxp3. In addition, their functional activity resembled that of Treg, as evidenced by a poor proliferative capacity; suppression of proliferation of CD25- CD4 or CD8T cells and inhibition of interferon-gamma release by CD25- CD4+ T cells. Reconstitution of Pan02-bearing Rag-/- mice with naive syngeneic CD25- CD4+ T cells induced CD25+ CD4+ Foxp3+ T cells in TDLNs, but not in the spleen. In contrast, Foxp3 was not detected in unreconstituted Pan02-bearing Rag-/- mice, or reconstituted mice bearing a TGF-beta-negative esophageal tumor. Furthermore, administration of neutralizing anti-TGF-beta antibody blocked the induction of Foxp3 in reconstituted Pan02-bearing Rag-/- mice. These results mimic earlier in vitro studies showing induction of Foxp3 through CD3 plus CD28 stimulation in the presence of TGF-beta. We conclude that Pan02 tumor promotes the prevalence of Treg, in part through the secretion of TGF-beta, which may result in immune evasion.

Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma.

Regulatory T cells (T(reg)) that prevent autoimmune diseases by suppression of self-reactive T cells may also suppress the immune response against cancer. In mice, depletion of T(reg) by Ab therapy leads to more efficient tumor rejection. T(reg)-mediated suppression of antitumor immune responses may partly explain the poor clinical response to vaccine-based immunotherapy for human cancer. In this study, we measured the prevalence of T(reg) that coexpress CD4 and CD25 in the PBLs, tumor-infiltrating lymphocytes, and regional lymph node lymphocytes from 65 patients with either pancreas or breast cancer. In breast cancer patients (n = 35), pancreas cancer patients (n = 30), and normal donors (n = 35), the prevalence of T(reg) were 16.6% (SE 1.22), 13.2% (SE 1.13), and 8.6% (SE 0.71) of the total CD4(+) cells, respectively. The prevalence of T(reg) were significantly higher in breast cancer patients (p < 0.01) and pancreas cancer patients (p < 0.01) when compared with normal donors. In tumor-infiltrating lymphocytes and lymph node lymphocytes, the T(reg) prevalence were 20.2% (SE 3.93) and 20.1% (SE 4.3), respectively. T(reg) constitutively coexpressed CTLA-4 and CD45RO markers, and secreted TGF-beta and IL-10 but did not secrete IFN-gamma. When cocultured with activated CD8(+) cells or CD4(+)25(-) cells, T(reg) potently suppressed their proliferation and secretion of IFN-gamma. We conclude that the prevalence of T(reg) is increased in the peripheral blood as well as in the tumor microenvironment of patients with invasive breast or pancreas cancers. These T(reg) may mitigate the immune response against cancer, and may partly explain the poor immune response against tumor Ags.