PPARγ inhibits breast cancer progression by upregulating PTPRF expression.

OBJECTIVE: Peroxisome proliferator-activated receptor γ (PPARγ) regulates fatty acid storage and glucose metabolism. Recently, PPARγ has been reported to be involved in cancer. The present study reported a PPARγ consensus binding site (AGGTCA) in the ptprf promoter and identified a strong association between PPARγ and PTPRF expression, as well as their tumor suppressor roles in a v-Ha-Ras-induced model of breast cancer. MATERIALS AND METHODS: The prognostic potential of PPARγ was assessed with a KM analysis of raw data from 3,951 breast cancer patients. The expression of PPARγ and PTPRF in the rat breast cancer cell lines was detected by Western blot and qPCR. The impact of PPARγ on cancer cell migration, invasion, and growth was confirmed using cell migration assay, transwell cell invasion assay, tri-dimensional soft agar culture, respectively. The binding of PPARγ with the ptprf promoter was then examined using electrophoretic mobility shift assay. The inhibitory effect of PPARγ on tumor growth was then examined in mouse tumor model in vivo. RESULTS: It was identified that PPARγ expression is lost in the aggressive v-Ha-Ras-induced breast cancer cell line FE1.2 but highly expressed in less malignant FE1.3 cells. Exogenous expression of PPARγ in FE1.2 cells (FE1.2-PPARγhi) resulted in a marked inhibition of proliferation compared with that in FE1.2-Vector control group. FE1.2-PPARγhi cells also exhibited reduced migration, invasion, and colony formation abilities compared with those of the controls. The PPARγ agonist rosiglitazone also suppressed the malignant properties of FE1.2 cells. Protein tyrosine phosphatase receptor F (PTPRF), a downstream target of PPARγ, was markedly induced in FE1.2-PPARγhi cells. A PPARγ consensus binding site (AGGTCA) was identified in the ptprf promoter, and an electrophoretic mobility shift assay confirmed that PPARγ bind to this promoter. Similar to the effect of vector-mediated overexpression of PPARγ, ectopic overexpression of PTPRF in FE1.2 cells led to reduced proliferation. Furthermore, a PPARγ antagonist (GW9662) and PTP inhibitor (NSC87877) abrogated the suppressive function of PPARγ and PTPRF in FE1.2 cells, respectively. PPARγ overexpression or activation suppressed the progression and distant organ metastasis of breast cancer cells in a NOD/SCID mouse model. CONCLUSIONS: These results suggest that PPARγ inhibits tumor cell proliferation, at least in part, through direct regulation of the ptprf gene and that PPARγ is a potential target for breast cancer treatment.

PPAR-delta modulates membrane cholesterol and cytokine signaling in malignant B cells.

A deeper understanding of the mechanisms that underlie aberrant signal transduction in B-cell cancers such as chronic lymphocytic leukemia (CLL) may reveal new treatment strategies. The lipid-activated nuclear receptor peroxisome proliferator-activated receptor delta (PPARδ) accounts for a number of properties of aggressive cancers and was found to enhance Janus kinase (JAK)-mediated phosphorylation of signal transducer and activator of transcription (STAT) proteins in B lymphoma cell lines and primary CLL cells. Autocrine production of cytokines such as IL10 and interferon-beta was not increased by PPARδ but signaling responses to these cytokines were amplified and associated with increased cholesterol biosynthesis and plasma membrane levels. Plasmalemmal cholesterol and STAT phosphorylation from type 1 interferons (IFNs) were increased by PPARδ agonists, transgenes and exogenous cholesterol, and decreased by cyclodextrin, PPARD deletion and chemical PPARδ inhibitors. Functional consequences of PPARδ-mediated perturbation of IFN signaling included impaired upregulation of co-stimulatory molecules. These observations suggest PPARδ modulates signaling processes in malignant B cells in part by altering cholesterol metabolism and changes the outcomes of signaling from cytokines such as IFNs. PPARδ antagonists may have therapeutic activity as anti-leukemic signal transduction modulators.

PPAR-delta promotes survival of chronic lymphocytic leukemia cells in energetically unfavorable conditions.

Targeting the mechanisms that allow chronic lymphocytic leukemia (CLL) cells to survive in harsh cancer microenvironments should improve patient outcomes. The nuclear receptor peroxisome proliferator activated receptor delta (PPARδ) sustains other cancers, and in silico analysis showed higher PPARD expression in CLL cells than normal lymphocytes and other hematologic cancers. A direct association was found between PPARδ protein levels in CLL cells and clinical score. Transgenic expression of PPARδ increased the growth and survival of CD5+ Daudi cells and primary CLL cells in stressful conditions including exhausted tissue culture media, low extracellular glucose, hypoxia and exposure to cytotoxic drugs. Glucocorticoids and synthetic PPARδ agonists up-regulated PPARD expression and also protected Daudi and primary CLL cells from metabolic stressors. Survival in low glucose was related to increased antioxidant expression, substrate utilization and mitochondrial performance, and was reversed by genetic deletion and synthetic PPARδ antagonists. These findings suggest PPARδ conditions CLL cells to survive in harsh microenvironmental conditions by reducing oxidative stress and increasing metabolic efficiency. Targeting PPARδ may be beneficial in the treatment of CLL.

PPAR-delta promotes survival of breast cancer cells in harsh metabolic conditions.

Expression of the nuclear receptor peroxisome proliferator activated receptor delta (PPARδ) in breast cancer cells is negatively associated with patient survival, but the underlying mechanisms are not clear. High PPARδ protein levels in rat breast adenocarcinomas were found to be associated with increased growth in soft agar and mice. Transgenic expression of PPARδ increased the ability of human breast cancer cell lines to migrate in vitro and form lung metastases in mice. PPARδ also conferred the ability to grow in exhausted tissue culture media and survive in low-glucose and other endoplasmic reticulum stress conditions such as hypoxia. Upregulation of PPARδ by glucocorticoids or synthetic agonists also protected human breast cancer cells from low glucose. Survival in low glucose was related to increased antioxidant defenses mediated in part by catalase and also to late AKT phosphorylation, which is associated with the prolonged glucose-deprivation response. Synthetic antagonists reversed the survival benefits conferred by PPARδ in vitro. These findings suggest that PPARδ conditions breast cancer cells to survive in harsh microenvironmental conditions by reducing oxidative stress and enhancing survival signaling responses. Drugs that target PPARδ may have a role in the treatment of breast cancer.

Prolonged clinical remissions in patients with relapsed or refractory follicular lymphoma treated with autologous stem cell transplantation incorporating rituximab.

Three sequential phase II trials were conducted with different immunotherapy approaches to enhance the outcome of autologous transplant (high-dose therapy and autologous stem cell transplantation (HDT/ASCT)) for recurrent follicular lymphoma. Seventy-three patients were enrolled from 1996 to 2009. Patients received HDT/ASCT combined with (1) interferon-α 3 MU/m(2) subcutaneously (SC) three times per week (TIW) for 2 years post-ASCT, (2) rituximab (R) 375 mg/m(2) for in vivo purging 3-5 days pre-stem cell collection and 2 × 4 weekly R at 2 and 6 months post-ASCT, respectively, or (3) three infusions of R pre-stem cell collection followed by 6× R weekly and interferon-α 3 MU/m(2) SC TIW. Although not statistically significant, progression-free survival (PFS) for patients who received rituximab was 56.4 and 49.1% at 5 and 10 years compared to 36 and 21% in those who did not receive rituximab. Molecular relapse post-HDT/ASCT was the strongest predictor of PFS in a multivariate analysis. Molecular relapse was coincident with or preceded clinical relapses in 84% of patients who relapsed­median of 12 months (range 0-129 months). Adverse events included secondary malignancy, transformation to diffuse large B cell lymphoma, prolonged mostly asymptomatic hypogammaglobulinemia, and pulmonary fibrosis. The long-term toxicity profile must be considered when selecting patients for this treatment.

A role for oleoylethanolamide in chronic lymphocytic leukemia.

Oleoylethanolamide (OEA) is a bioactive lipid that stimulates nuclear and G protein-coupled receptors and regulates appetite and fat metabolism. It has not previously been shown to have a role in cancer. However, a mass spectrometry-based lipidomics platform revealed the presence of high amounts of OEA in the plasma of chronic lymphocytic leukemia (CLL) patients compared with normal donors. CLL cells produced OEA and the magnitude of plasma OEA levels was related directly to the circulating leukemic cell number. OEA from CLL cells was increased by URB-597, an inhibitor of fatty acid amide hydrolase (FAAH), and decreased by inflammatory mediators that downregulate expression of N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD). These enzymes degrade and synthesize OEA, respectively. Nonphysiologic doses of OEA prevented spontaneous apoptosis of CLL cells in a receptor-independent manner that was mimicked by its free fatty acid (FFA) derivative oleate. However, OEA-containing supernatants from CLL cells induced lipolysis in adipocytes, lipid products from adipocytes protected CLL cells from cytotoxic chemotherapy, and increased levels of FFAs were found in CLL plasma that correlated with OEA. We suggest OEA is a lipolytic factor produced by CLL cells to fuel their growth with a potential role in drug resistance and cancer cachexia.

PPAR-alpha is a therapeutic target for chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) cells with aggressive clinical properties express lipoprotein lipase (LPL), which generates activating ligands for the nuclear receptor peroxisome proliferator activated receptor (PPAR)α and allows fatty acids to be used as fuel. However, the role of PPARα in CLL is unclear. PPARα was found to be expressed by circulating CLL cells and highly associated with advanced stage disease. Consistent with this observation, palmitate oxidation rates in circulating CLL cells were similar to more conventional fat-burning cells such as muscle. Transgenic expression of PPARα in CD5(+) Daudi cells increased both their expression of immunosuppressive factors (that is, interleukin (IL)10 and phospho-STAT3) and resistance to metabolic and cytotoxic stressors. In contrast, marked downregulation of PPARα expression accompanied immunogenic death of proliferating CLL cells. The PPARα antagonist MK886 killed circulating CLL cells directly, caused proliferating CLL cells to enter an immunogenic death pathway and cleared CLL xenografts from immunodeficient mice. These results suggest that PPARα is a biological mediator of CLL and MK886 is a clinically relevant agent with activity against CLL.

Differential immune responses mediated by adenovirus- and lentivirus-transduced DCs in a HER-2/neu overexpressing tumor model.

Recent investigations have demonstrated that adenoviral and lentiviral vectors encoding HER-2 can be utilized in cancer immunotherapy. However, it is not known whether both viral systems elicit a similar immune response. Here, we compare the immune response in mice induced by dendritic cells (DCs) infected with either recombinant adenovirus or lentivirus encoding rat HER-2 (rHER-2). Both vaccine types yielded similar control of tumor growth, but we found clear differences in their immune responses 10 days after DC immunization. Adenovirus rHER-2-transduced DCs elicited locally and systemically high frequencies of CD4+ and CD8+ T cells, while lentivirus rHER-2-transduced DCs predominantly led to CD4+ T-cell infiltration at the tumor site. Splenocytes from mice immunized with lentivirus rHER-2-transduced DCs secreted higher levels of interferon (IFN)-γ, mainly by CD4+ T cells, following stimulation by RM-1-mHER-2 tumors. In contrast, the adenovirus vaccinated group exhibited CD4+ and CD8+ T cells that both contributed to IFN-γ production. Besides an established cellular immune response, the rHER-2/DC vaccine elicited a significant humoral response that was highest in the adenovirus group. DC subsets and regulatory T cells in the spleen were also differentially modulated in the two vaccine systems. Finally, adoptive transfer of splenocytes from both groups of immunized mice strongly inhibited in vivo tumor growth. Our results suggest that not only the target antigen but also the virus system may determine the nature and magnitude of antitumor immunity by DC vaccination.

Aberrant O-GlcNAcylation characterizes chronic lymphocytic leukemia.

O-linked N-Acetylglucosamine (O-GlcNAc) post-translational modifications originate from the activity of the hexosamine pathway, and are known to affect intracellular signaling processes. As aberrant responses to microenvironmental signals are a feature of chronic lymphocytic leukemia (CLL), O-GlcNAcylated protein levels were measured in primary CLL cells. In contrast to normal circulating and tonsillar B cells, CLL cells expressed high levels of O-GlcNAcylated proteins, including p53, c-myc and Akt. O-GlcNAcylation in CLL cells increased following activation with cytokines and through toll-like receptors (TLRs), or after loading with hexosamine pathway substrates. However, high baseline O-GlcNAc levels were associated with impaired signaling responses to TLR agonists, chemotherapeutic agents, B cell receptor crosslinking and mitogens. Indolent and aggressive clinical behavior of CLL cells were found to correlate with higher and lower O-GlcNAc levels, respectively. These findings suggest that intracellular O-GlcNAcylation is associated with the pathogenesis of CLL, which could potentially have therapeutic implications.

Prolonging microtubule dysruption enhances the immunogenicity of chronic lymphocytic leukaemia cells.

Cytotoxic chemotherapies do not usually mediate the expression of an immunogenic gene programme in tumours, despite activating many of the signalling pathways employed by highly immunogenic cells. Concomitant use of agents that modulate and complement stress-signalling pathways activated by chemotherapeutic agents may then enhance the immunogenicity of cancer cells, increase their susceptibility to T cell-mediated controls and lead to higher clinical remission rates. Consistent with this hypothesis, the microtubule inhibitor, vincristine, caused chronic lymphocytic leukaemia (CLL) cells to die rapidly, without increasing their immunogenicity. Protein kinase C (PKC) agonists (such as bryostatin) delayed the death of vincristine-treated CLL cells and made them highly immunogenic, with increased stimulatory abilities in mixed lymphocyte responses, production of proinflammatory cytokines, expression of co-stimulatory molecules and activation of c-Jun N-terminal kinase (JNK), p38 and nuclear factor kappa B (NF-kappaB) signalling pathways. This phenotype was similar to the result of activating CLL cells through Toll-like receptors (TLRs), which communicate 'danger' signals from infectious pathogens. Use of PKC agonists and microtubule inhibitors to mimic TLR-signalling, and increase the immunogenicity of CLL cells, has implications for the design of chemo-immunotherapeutic strategies.

Rituximab purging and maintenance combined with auto-SCT: long-term molecular remissions and prolonged hypogammaglobulinemia in relapsed follicular lymphoma.

We enrolled 23 patients with relapsed follicular lymphoma (FL) in a prospective single-arm study of auto-SCT combined with in vivo rituximab graft purging and post transplant rituximab maintenance. Minimal residual disease was monitored with quantitative PCR testing. With a median follow-up of 74.2 months, neither median overall survival (OS) nor PFS has been reached. Here, 5-year OS and 5-year PFS are 78% (95% confidence interval (CI) 61-95%) and 59% (95% CI 38-80%), respectively. Time to progression (TTP) with the experimental regimen was significantly improved compared with TTP with the last prior treatment (P<0.001). Durable molecular remissions occurred in 11 of 13 assessable patients. PFS was significantly longer in patients who achieved a molecular remission by 3 months post-auto-SCT (P=0.001). Prolonged hypogammaglobulinemia occurred in most patients; however, no increase in major infections was observed.

Obstacles to effective Toll-like receptor agonist therapy for hematologic malignancies.

It has long been noted that products of microorganisms have clinical activity against hematologic malignancies. Recent advances suggest that Toll-like receptors (TLRs) activated by ligands in the microbial preparations might account for some of this activity, and that defined TLR agonists might improve the clinical efficacy of this approach. A potentially important mechanism of action of TLR agonists is their ability to cause tumor cells to differentiate into a 'tolerized' state in which they become highly sensitive to cytotoxic effector cells and chemotherapeutic drugs. TLR agonists as single agents have strong activity against cutaneous leukemias and lymphomas but are not as effective against systemic disease. A possible reason for this discrepancy is the hypoxic internal tumor microenvironment, which promotes glycolytic metabolism, and the presence of suppressive cytokines, prostaglandins and nucleosides that prevent strong TLR signaling in cancer cells. Accordingly, concomitant use of agents to counter this intrinsic microenvironmental inhibition, together with TLR agonists, may prove to be an effective treatment strategy for the hematologic malignancies.

Toll-like receptor agonists in the treatment of chronic lymphocytic leukemia.

Advances in our understanding of the Toll-like receptors (TLRs) have led to the identification of several agonists that are suitable for clinical development. Chronic lymphocytic leukemia (CLL) may be especially amenable to TLR agonists because it is an immunologically susceptible tumor with strong expression of several TLRs, particularly TLR-7 and TLR-9. TLR agonists may indirectly clear CLL cells by enhancing the activity of natural killer and tumor-reactive T cells, or by altering the tumor microenvironment and inhibiting angiogenesis. However, signaling pathways can be activated directly in CLL cells by TLR-7 and TLR-9 agonists, leading to the production of cytokines and costimulatory molecules in a manner that is dependent on the underlying cytogenetic abnormalities, but rendering the tumor cells more sensitive to killing by cytotoxic T cells, immunotoxins and some chemotherapeutic drugs. Imidazoquinolines are TLR-7 agonists with strong local activity against CLL, and phase I trials of systemically administered imidazoquinolines (and also cytosine-phosphate-guanosine oligonucleotides that are TLR-9 agonists) are currently ongoing at different centers. The potential importance of these TLR agonists in the treatment of CLL is suggested by their ability to sensitize tumor cells to cytotoxic agents, and their future probably lies in combination with radiotherapies, chemotherapies, monoclonal antibodies and cancer vaccines.

Immunomodulatory effects of Toll-like receptor-7 activation on chronic lymphocytic leukemia cells.

Weak immunogenicity of chronic lymphocytic leukemia (CLL) cells may contribute to disease progression and inhibit effective immunotherapy. Accordingly, agents that enhance the immunogenicity of CLL cells may be useful in immunotherapeutic approaches to this disease. Since Toll-like receptors (TLRs) are major regulators of innate immunity and initiation of adaptive immunity, we studied the effects of viral pathogen associated molecular pattern agonists (that are recognized by TLRs) on the costimulatory phenotype and function of CLL cells. CLL cells (especially those with high endogenous expression of CD38) responded to TLR7-activating imidazoquinolines and guanosine analogs by increasing costimulatory molecule expression, producing inflammatory cytokines, and becoming more sensitive to killing by cytotoxic effectors. Additional activation of protein kinase C pathways increased the ability to stimulate T-cell proliferation, blocked phosphorylation of the transcription factor, signal transducer and activator of transcription (STAT)3, and resulted in the acquisition of a dendritic cell surface phenotype by TLR7-activated CLL cells. Normal B cells also responded to TLR7 activation by increasing costimulatory molecule expression and cytokine production. These findings suggest a potential role for TLR7 agonists in CLL immunotherapy.

Bone marrow aspirates as part of routine donor assessment for allogeneic blood and marrow transplantation can reveal presence of occult hematological malignancies in otherwise asymptomatic individuals.

Pre transplant screening work-up of donors for allogeneic blood and marrow transplantation is essential in an effort to minimize risks to the recipient and protect the donor. At Princess Margaret Hospital, every potential donor is screened with a bone marrow aspirate. The case histories of three asymptomatic potential donors who presented within 1 year with normal complete blood counts, history and physical examination are presented. A 65-year-old male patient was diagnosed with smouldering multiple myeloma, a 72-year-old male patient with chronic lymphocytic leukemia and a 42-year-old male patient with myelodysplastic syndrome. Bone marrow examination led to the diagnosis in each one of these cases. Of note is that each of the potential donors was discovered to have the same disease as the transplant recipient. In vitro clonogenic hemopoietic progenitor assays were compared to those of 20 normal volunteers. Inferior growth of hemopoietic progenitor colonies in all three was noted. In conclusion, particularly in older donors and donors with potential for familial malignancies, more screening investigations including bone marrow aspiration may be reasonable to investigate for occult hematological malignancies prior to stem cell donation. Clonogenic assays can contribute to detect hemopoietic abnormalities pre transplant.