Debra Mikkola

Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration

A shot in the arm for damaged tissue Tissue damage can be caused by injury, disease, and even certain medical treatments. There is great interest in identifying drugs that accelerate tissue regeneration and recovery, especially drugs that might benefit multiple organ systems. Zhang et al. describe a compound with this desired activity, at least in mice (see the Perspective by FitzGerald). SW033291 promotes recovery of the hematopoietic system after bone marrow transplantation, prevents the development of ulcerative colitis in the intestine, and accelerates liver regeneration after hepatic surgery. It acts by inhibiting an enzyme that degrades prostaglandins, lipid signaling molecules that have been implicated in tissue stem cell maintenance. Science, this issue 10.1126/science.aaa2340; see also p. 1208 A compound that inhibits prostaglandin degradation enhances tissue regeneration in multiple organs in mice. [Also see Perspective by FitzGerald] INTRODUCTION Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. To date, therapeutic interventions have largely focused on targeting two PGE2 biosynthetic enzymes, cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2), with the aim of reducing PGE2 production. In this study, we take the converse approach: We examine the role of a prostaglandin-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), as a negative regulator of tissue repair, and we explore whether inhibition of this enzyme can potentiate tissue regeneration in mouse models. RATIONALE We used 15-PGDH knockout mice to elucidate the role of 15-PGDH in regulating tissue levels of PGE2 and tissue repair capacity in multiple organs. We then developed SW033291, a potent small-molecule inhibitor of 15-PGDH with activity in vivo. We used SW033291 to investigate the therapeutic potential of 15-PGDH inhibitors in tissue regeneration and to identify a 15-PGDH–regulated hematopoietic pathway within the bone marrow niche. RESULTS We found that in comparison with wild-type mice, 15-PGDH–deficient mice display a twofold increase in PGE2 levels across multiple tissues—including bone marrow, colon, and liver—and that they show increased fitness of these tissues in response to damage. The mutant mice also show enhanced hematopoietic capacity, with increased neutrophils, increased bone marrow SKL (Sca-1+ C-kit+ Lin−) cells (enriched for stem cells), and greater capacity to generate erythroid and myeloid colonies in cell culture. The 15-PGDH–deficient mice respond to colon injury from dextran sulfate sodium (DSS) with a twofold increase in cell proliferation in colon crypts, which confers resistance to DSS-induced colitis. The mutant mice also respond to partial hepatectomy with a greater than twofold increase in hepatocyte proliferation, which leads to accelerated and more extensive liver regeneration. SW033291, a potent small-molecule inhibitor of 15-PGDH (inhibitor dissociation constant Ki ~0.1 nM), recapitulates in mice the phenotypes of 15-PGDH gene knockout, inducing increased hematopoiesis, resistance to DSS colitis, and more rapid liver regeneration after partial hepatectomy. Moreover, SW033291-treated mice show a 6-day-faster reconstitution of hematopoiesis after bone marrow transplantation, with accelerated recovery of neutrophils, platelets, and erythrocytes, and greater recovery of bone marrow SKL cells. This effect is mediated by bone marrow CD45– cells, which respond to increased PGE2 with a fourfold increase in production of CXCL12 and SCF, two cytokines that play key roles in hematopoietic stem cell homing and maintenance. CONCLUSIONS Studying mouse models, we have shown that 15-PGDH negatively regulates tissue regeneration and repair in the bone marrow, colon, and liver. Of most direct utility, our observations identify 15-PGDH as a therapeutic target and provide a chemical formulation, SW033291, that is an active 15-PGDH inhibitor in vivo and that potentiates repair in multiple tissues. SW033291 or related compounds may merit clinical investigation as a strategy to accelerate recovery after bone marrow transplantation and other tissue injuries. Inhibiting 15-PGDH accelerates tissue repair. (A) The enzyme 15-PGDH degrades and negatively regulates PGE2. (B) SW033291 inhibits 15-PGDH, increases tissue levels of PGE2, and induces CXCL12 and SCF expression from CD45– bone marrow cells. This in turn accelerates homing of transplanted hematopoietic stem cells (HSC), generation of mature blood elements, and post-transplant recovery of normal blood counts. Inhibiting 15-PGDH similarly stimulates cell proliferation after injury to colon or liver, accelerating repair of these tissues. Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. Here, we show that inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme, potentiates tissue regeneration in multiple organs in mice. In a chemical screen, we identify a small-molecule inhibitor of 15-PGDH (SW033291) that increases prostaglandin PGE2 levels in bone marrow and other tissues. SW033291 accelerates hematopoietic recovery in mice receiving a bone marrow transplant. The same compound also promotes tissue regeneration in mouse models of colon and liver injury. Tissues from 15-PGDH knockout mice demonstrate similar increased regenerative capacity. Thus, 15-PGDH inhibition may be a valuable therapeutic strategy for tissue regeneration in diverse clinical contexts.

TGF- β -induced nuclear localization of Smad2 and Smad3 in Smad4 null cancer cell lines

Smad4 is a tumor suppressor gene that is commonly lost or mutated in colorectal and pancreatic cancers. The activated transforming growth factor-β (TGF-β) receptor phosphorylates Smad2 and Smad3, which then complex with Smad4 and translocate to the nucleus. Smad4 mutations when detected as present in some human cancers have been considered sufficient to inactivate TGF-β signaling. In this work, we describe a colon cancer cell line, VACO-9M, that is Smad4 null when analysed by multiple assays. To study the role of Smad4 in TGF-β-induced translocation of the receptor-activated Smads to the nucleus, we analysed by immunofluorescence the cellular localization of endogenous Smad2 and Smad3 after TGF-β treatment of VACO-9M, plus four additional Smad4 null cell lines of breast (MDA-MB-468), or pancreatic (BxPC3, Hs766T, CFPAC-1) origin. In each cell line, TGF-β treatment resulted in both Smad2 and Smad3 moving to the nucleus in a Smad4-independent fashion. Nuclear translocation of Smad2 and Smad3 was, however, not sufficient to activate reporters for TGF-β-induced transcriptional responses, which were however restored by transient transfection of wild-type Smad4. We conclude that Smad4 is not required for nuclear translocation of Smad2 and Smad3, but is needed activation of at least certain transcriptional responses.

Detection of clonally restricted immunoglobulin heavy chain gene rearrangements in normal and lesional skin: analysis of the B cell component of the skin-associated lymphoid tissue and implications for the molecular diagnosis of cutaneous B cell lymphomas.

A monoclonal B cell population is the hallmark of B cell neoplasms including cutaneous B cell lymphomas (CBCLs). We modified and tested several polymerase chain reaction (PCR)-based assays involving amplification of immunoglobulin heavy chain (IgH) gene rearrangements to optimize assays specifically for cutaneous lymphoid infiltrates. We achieved greatest sensitivity with an assay employing IgH consensus primers complementary to the framework 3 portion of the upstream variable region and the downstream joining region. We studied 12 CBCLs, 6 nodal lymphomas and 7 cell lines. In 17/25 of these B cell neoplasms (84%), we detected one or two dominant bands, consistent with one or both IgH alleles being rearranged in the neoplastic B cell clone. As expected, IgH PCR assays produced diffuse smears in agarose gels or complex ladders in polyacrylamide gels when polyclonal B cell controls (blood and tonsil) were analyzed. However, in normal skin and non-CBCL skin lesions, one or a small number of discrete bands were sometimes detected. In certain cases, this made it difficult to distinguish true positives (monoclonal CBCL) from false positives (clonally restricted benign B cells). Correlation with immunophenotyping confirmed that false positive results were confined to samples with sparse or immunohistologically undetectable B cell infiltrates. Pseudoclonal bands showed variable sizes in repeat PCR reactions and could be distinguished from monoclonal bands by polyacrylamide gel electrophoresis of pooled triplicate PCR products. These findings suggest that molecular diagnosis using IgH PCR assays is best suited for B-cell-rich infiltrates, and can be problematic when applied to suspected T-cell-rich CBCLs, cutaneous T cell lymphomas, or other lesions containing only few B cells unless one is cognizant of the potential pitfalls. Furthermore, these results demonstrate the presence of rare B cells in normal skin and immunohistologically defined cutaneous T cell infiltrates. This correlates with recent reports of sparse B cells within the lymph draining from normal skin and may represent molecular evidence for a trafficking B cell component of the skin-associated lymphoid tissue (SALT). It also suggests a candidate B cell subset for the pathogenesis of cutaneous lymphoid hyperplasia and CBCLs.

Solitary primary cutaneous CD30+ large cell lymphoma of natural killer cell phenotype bearing the t(2;5)(p23;q35) translocation and presenting in a child

Primary cutaneous CD30+ large cell lymphoma is an unusual tumor most commonly seen in adults. Most of these lymphomas are of T-cell origin and carry a good prognosis. We present the case of a 4-year-old girl with stage IEA CD30+ large cell lymphoma with a CD56+ natural killer cell phenotype and the t(2;5)(p23;q35) translocation. After excision, the patient has been free of disease for 44 months. Primary cutaneous CD30+ large cell lymphoma is uncommon in children. To our knowledge, primary cutaneous CD30+ natural killer type lymphoma has not been reported previously. The indolent behavior of this tumor indicates its similarity to other primary cutaneous CD30+ large cell lymphomas and its difference from other CD56+ lymphomas involving the skin, which often exhibit an aggressive clinical course. Cases such as this one illustrate why the use of a single, or even a few, immunohistochemical stains can be misleading in regard to lymphoma classification and prognostication.

Induction of KIAA1199/CEMIP is associated with colon cancer phenotype and poor patient survival

Genes induced in colon cancer provide novel candidate biomarkers of tumor phenotype and aggressiveness. We originally identified KIAA1199 (now officially called CEMIP) as a transcript highly induced in colon cancer: initially designating the transcript as Colon Cancer Secreted Protein 1. We molecularly characterized CEMIP expression both at the mRNA and protein level and found it is a secreted protein induced an average of 54-fold in colon cancer. Knockout of CEMIPreduced the ability of human colon cancer cells to form xenograft tumors in athymic mice. Tumors that did grow had increased deposition of hyaluronan, linking CEMIP participation in hyaluronan degradation to the modulation of tumor phenotype. We find CEMIP mRNA overexpression correlates with poorer patient survival. In stage III only (n = 31) or in combined stage II plus stage III colon cancer cases (n = 73), 5-year overall survival was significantly better (p = 0.004 and p = 0.0003, respectively) among patients with low CEMIP expressing tumors than those with high CEMIP expressing tumors. These results demonstrate that CEMIP directly facilitates colon tumor growth, and high CEMIP expression correlates with poor outcome in stage III and in stages II+III combined cohorts. We present CEMIP as a candidate prognostic marker for colon cancer and a potential therapeutic target.

The clonality of tumor-infiltrating lymphocytes in African Kaposi's sarcoma.

Background: African Kaposi’s sarcoma (KS) lesions contain human herpesvirus‐8 (HHV‐8) and Epstein‐Barr virus (EBV), both of which are associated with various types of non‐Hodgkin’s lymphomas and are known to produce several factors suspected of lymphomagenic potential. The aim of this study was to evaluate tumor‐infiltrating lymphocytes for the evidence of clonal expansion in African KS.

Abstract 3126: CEMIP, a secreted protein highly induced in colon cancer and associated with poor patient survival

Background: Colorectal cancer is the second leading cause of cancer death among adult Americans. Tumor stage still remains the clinical standard for determining prognosis of colon cancer patients and for selecting individuals for treatment with adjuvant chemotherapy. Genes induced in colon cancer provide novel candidate biomarkers of tumor phenotype and aggressiveness. The goal of this study was to identify genes whose expression is dramatically up-regulated in colon neoplasia even at the early stages of the disease, and with the potential to be new prognostic markers of patient outcome and/or targets for new therapies. Methods: We used expression microarrays, real-time PCR, Western blot, and immunohistochemistry to identify CEMIP (originally named KIAA1199/CCSP1) induction in colon cancer, and characterized the biological properties of the corresponding protein in cell-line and mouse xenografts. Sandwich ELISA was developed to determine CEMIP plasma levels in patients with colon cancer. Prognostic importance of gene induction was demonstrated by real-time PCR measurement of gene expression in colon cancer cases of known clinical outcome. Results: We originally identified CEMIP as a novel transcript that is induced an average of 54-fold in colon cancer, with a similar increase in protein level. We find that CEMIP is a secreted protein and that plasma levels of CEMIP in colon cancer patients is increased compared to normal subjects (P = 0.05). Knocking out CEMIP in a human colon cancer cell line markedly reduced growth of tumor xenografts implanted in athymic mice. Tumors that did grow had increased deposition of hyaluronan, linking CEMIP participation in hyaluronan degradation to the modulation of tumor phenotype. Human stage III colon cancer cases with greater than average increased tumor CEMIP expression had a median survival time of 37 months, versus greater than 140 months for colon cancer cases with below average CEMIP expression (P = 0.004). Similarly, among combined stage II plus III colon cancer cases, median survival decreased by 92 months for CEMIP high versus low expressing tumors (P = 0.0003). Conclusions: CEMIP is highly expressed in colon neoplasia and is a novel member of the colon cancer secreted proteome making it a candidate serological marker of early human colon neoplasia. CEMIP facilitates tumor growth, and high CEMIP correlates with poor outcome in stage III and in stages II plus III combined cohorts. CEMIP may have utility as both a prognostic marker of colon cancer outcome, and as a potential therapeutic target. Citation Format: Stephen P. Fink, Lois Myeroff, Revital Kariv, Petra Platzer, Baozhong Xin, Debra Mikkola, Earl Lawrence, Nathan Morris, Arman Nosrati, James Willson, Joseph Willis, Martina Veigl, Jill Barnholtz-Sloan, Zhenghe Wang, Sanford Markowitz. CEMIP, a secreted protein highly induced in colon cancer and associated with poor patient survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3126.