Role of Roflumilast Combined with ESHAP Chemotherapy in Relapsed/Refractory Patients with Diffuse Large B-Cell Lymphoma.

PURPOSE: There are unmet needs associated with the current treatment strategies for relapsed/refractory diffuse large B-cell lymphoma (DLBCL) due to the poor treatment outcomes of these strategies. Roflumilast, a selective phosphodiesterase-4 inhibitor used for treating chronic obstructive pulmonary disease, is effective against B-cell malignancy via phosphoinositide 3-kinase (PI3K)-activity suppression. We analyzed the effects of roflumilast combined with ESHAP (etoposide, cisplatin, methylprednisolone, and cytarabine) chemotherapy in experimental and clinical settings. MATERIALS AND METHODS: An in vitro study using lymphoma cell lines and a pilot study on relapsed/refractory DLBCL patients were conducted to investigate the effects and mechanism of the combination of roflumilast and chemotherapy. The complete response (CR), overall response rate (ORR), and 1-year progression-free survival (PFS) were analyzed. RESULTS: We found that roflumilast is efficient when combined with other chemotherapy drugs, especially cytarabine. Synergistic effects between these two drugs influence the translation of mammalian target of rapamycin and myeloid cell leukemia 1, resulting in apoptosis and inhibition of B-cell lymphoma proliferation. In clinical setting, the roflumilast group showed better rates of CR (46.2% vs. 34.6%), ORR (76.9% vs. 53.8%), and 1-year PFS (50.0% vs. 25.9%) compared with the control group, though not statistically significant. The roflumilast group showed a higher incidence of asthenia and gastrointestinal adverse events. However, grade 3 or 4 adverse events were similar in both groups. CONCLUSION: We found that roflumilast, when combined with ESHAP chemotherapy, for relapsed/refractory DLBCL was clinically active and well tolerated. This combined treatment was able to suppress PI3K activity, which is correlated with the degree of clinical response.

Predictive Parameters of Febrile Neutropenia and Clinical Significance of G-CSF Receptor Signaling Pathway in the Development of Neutropenia during R-CHOP Chemotherapy with Prophylactic Pegfilgrastim in Patients with Diffuse Large B-Cell Lymphoma.

PURPOSE: Pegfilgrastim is widely used to prevent chemotherapy-induced neutropenia (CIN) and febrile neutropenia (FN) in patients with diffuse large B-cell lymphoma (DLBCL). We investigated the predictive factors affecting CIN and FN incidence in patients with DLBCL receiving rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) chemotherapy with pegfilgrastim and conducted experiments to find reason for the occurrence of CIN even when pegfilgrastim was used. MATERIALS AND METHODS: We reviewed the CIN and FN events of 200 patients with DLBCL. Based on these data, we investigate the association with predictive factor and the levels of granulocyte-colony stimulating factor (G-CSF) receptor signaling pathway markers (pSTAT3, pAKT, pERK1/2, pBAD, and CXCR4) in bone marrow (BM) samples isolated from patients with DLBCL. RESULTS: FN was significantly associated with stage III/IV (hazard ratio [HR], 12.74) and low serum albumin levels (HR, 3.87). Additionally, patients with FN had lower progression-free survival (PFS; 2-year PFS, 51.1 % vs. 74.0%) and overall survival (OS; 2-year OS, 58.2% vs. 85.0%) compared to those without FN. The occurrence of CIN was associated with overexpression of G-CSF receptor signaling pathway markers, and expression levels of these markers were upregulated in BM cells co-cultured with DLBCL cells. The rate of neutrophil apoptosis was also higher in neutrophils co-cultured with DLBCL cells and was further promoted by treatment with doxorubicin. CONCLUSION: Our findings suggest that high DLBCL burden may alter the BM environment and G-CSF receptor signaling pathway, even in chemotherapy-naïve state, which may increase CIN frequency during R-CHOP chemotherapy.

NF-κB p65 represses microRNA-124 transcription in diffuse large B-cell lymphoma.

BACKGROUND: Previous studies have shown that the copy number of microRNA (miR)-124 is decreased in diffuse large B cell lymphoma (DLBCL), and that miR-124 is a tumor suppressor by targeting NF-κB p65 in B-cell lymphoma. In turn, miR-124 expression is regulated by transcription factors such as HNF4α, ETS2, and p53. However, whether and how miR-124 transcription is modulated by NF-κB transcription factors remain unknown in DLBCL. OBJECTIVE: To investigate whether the activation of NF-κB signaling could inhibit the expression of miR-124, possibly contributing to the pathogenesis of DLBCL. METHODS: Potential transcription factors regulating miR-124 transcription were predicted using the Transfac software. The cellular effects of NF-κB p65 on miR-124 were examined by MTS assay, Western blot assay, qPCR, and chromatin immunoprecipitation (ChIP) assays using DLBCL cell lines. RESULTS: Inhibition of NF-κB signals using Bay11-7085 increased miR-124 expression whereas exposure to TNF-α decreased it. Ectopic expression of p65 suppressed miR-124 expression, suggesting that p65 may be a transcriptional repressor of miRNA-124. Pharmacological analyses showed that phosphorylated/activated p65 downregulates miR-124 via two signaling pathways: (1) TAK1/IKKα-IKKß/IκBα and (2) MAPK/p65. Moreover, ChIP assay demonstrated that p65 directly regulates miR-124 by binding to the NF-κB consensus sequence in its promoter region. Finally, we also confirmed that stable ectopic expression of miR-124 suppresses cell proliferation and survival. CONCLUSION: Taken together, our study uncovered a mechanism by which active NF-κB signaling disrupts the function of miR-124 as a tumor suppressor in DLBCL.

Inhibition of phosphodiesterase 4D decreases the malignant properties of DLD-1 colorectal cancer cells by repressing the AKT/mTOR/Myc signaling pathway.

Colorectal cancer (CRC) is a complex disease involving numerous genetic abnormalities. One of the major characteristics of CRC is enhanced Wnt signaling caused by loss-of-function mutations in the adenomatous polyposis coli (APC) gene. Previously, it has been demonstrated that the majority of malignant phenotypes following APC deletion in adult murine small intestines could be rescued when Myc, a downstream target of the Wnt pathway, was deleted. This indicated that Myc is a critical regulator of CRC development following APC loss. Previous studies reported that cyclic adenosine 3',5'-monophosphate (cAMP) can influence the AKT/mammalian target of rapamycin (mTOR) survival pathway in cancer and Myc is a critical downstream molecule of AKT/mTOR signaling. Phosphodiesterase 4D (PDE4D), a member of the cAMP-specific PDE4 family, has been associated with drug resistance in CRC. However, the association between PDE4D and Myc remains unclear. To investigate the potential role of PDE4D in Myc regulation in CRC, the present study evaluated the expression levels of PDE4 subtypes in DLD-1 CRC cells. Additionally, the effects of PDE4 inhibitors on Myc expression and oncogenic properties were analyzed by western blot analysis, reverse transcription-quantitative polymerase chain reaction, colony formation and soft agar assays. It was demonstrated that cAMP/PDE4D signals serve a critical role in regulating Myc expression in DLD-1 CRC cells. Furthermore, PDE4D was identified to be a main hydrolyzer of cAMP and suppression of PDE4D using selective inhibitors of PDE4 increased intracellular cAMP levels, which resulted in a marked decrease in the oncogenic properties of DLD-1 cells, including colony formation, cell proliferation and anchorage-independent growth. Notably, the current data imply that cAMP represses Myc expression via the downregulation of AKT/mTOR signaling, which was abolished by high PDE4D activities in DLD-1 cells. Additionally, a natural polyphenol resveratrol in combination with forskolin elevated the concentration of cAMP and enhanced the expression of Myc and the malignant phenotype of DLD-1 cells, reproducing the effect of known chemical inhibitors of PDE4. In conclusion, the present study identified that cAMP/PDE4D signaling is a critical regulator of Myc expression in DLD-1 and possibly other CRC cells.

Disruption of the Myc-PDE4B regulatory circuitry impairs B-cell lymphoma survival.

A large body of evidence suggests that B-cell lymphomas with enhanced Myc expression are associated with an aggressive phenotype and poor prognosis, which makes Myc a compelling therapeutic target. Phosphodiesterase 4B (PDE4B), a main hydrolyzer of cyclic AMP (cAMP) in B cells, was shown to be involved in cell survival and drug resistance in diffuse large B cell lymphomas (DLBCL). However, the interrelationship between Myc and PDE4B remains unclear. Here, we first demonstrate the presence of the Myc-PDE4B feed-forward loop, in which Myc and PDE4B mutually reinforce the expression of each other. Next, the combined targeting of Myc and PDE4 synergistically prevented the proliferation and survival of B lymphoma cells in vitro and in a mouse xenograft model. We finally recapitulated this combinatorial effect in Eµ-myc transgenic mice; co-inhibition of Myc and PDE4 suppressed lymphomagenesis and restored B cell development to the wild type level that was associated with marked reduction in Myc levels, unveiling the critical role of the Myc-PDE4B amplification loop in the regulation of Myc expression and the pathogenesis of B cell lymphoma. These findings suggest that the disruption of the Myc-PDE4B circuitry can be exploited in the treatment of B cell malignancies.

Angelica gigas Nakai and Decursin Downregulate Myc Expression to Promote Cell Death in B-cell Lymphoma.

Angelica gigas Nakai (AGN) is an oriental traditional medicine to treat anemia, dysmenorrhea, and migraine. However, its anti-lymphoma effect is yet to be tested. Here, we demonstrated that AGN and its major component decursin target Myc to suppress lymphomagenesis in vitro and in vivo. AGN inhibited cell viability in multiple B lymphoma cells, while sparing normal splenocytes and bone marrow cells. Increased cleaved PARP level and caspase 3/7 activity and the repression of survival-promoting AKT/mTOR and MAPK pathways downstream of BCR, were responsible for the pro-apoptotic effects of AGN. We found that Myc, a prominent downstream target of these signaling pathways, contributes to AGN-induced cell death. Moreover, co-treatment with AGN and a Myc inhibitor, JQ1 or 10058-F4 yielded synergistic cytotoxic activities against cancer cells with markedly reduced Myc expression. AGN downregulated Myc expression and suppressed tumorigenesis in Eµ-myc transgenic mice. The proapoptotic activities of AGN were recapitulated by decursin, indicating that the anti-tumor effect of AGN was mainly caused by decursin. These findings suggest that AGN and decursin possess potent anti-lymphoma activity, and combination therapies with AGN/decursin and a Myc inhibitor to target Myc more efficiently could be a valuable avenue to explore in the treatment of B-cell lymphoma.

Inhibition of c-FLIPL expression by miRNA-708 increases the sensitivity of renal cancer cells to anti-cancer drugs.

Dysregulation of the anti-apoptotic protein, cellular FLICE-like inhibitory protein (c-FLIP), has been associated with tumorigenesis and chemoresistance in various human cancers. Therefore, c-FLIP is an excellent target for therapeutic intervention. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in tumorigenesis, tumor suppression, and resistance or sensitivity to anti-cancer drugs. However, whether miRNAs can suppress c-FLIPL expression in cancer cells is unclear. The aim of this study was to identify miRNAs that could inhibit the growth of renal cancer cells and induce cell death by inhibiting c-FLIPL expression. We found that MiRNA-708 and c-FLIPL expression were inversely correlated. While c-FLIPL expression was upregulated, miRNA-708 was rarely expressed in renal cancer cells. Luciferase reporter assays demonstrated that miRNA-708 negatively regulated c-FLIPL expression by binding to the miRNA-708 binding site in the 3' untranslated region (3'UTR) of c-FLIPL. Ectopic expression of miRNA-708 increased the accumulation of sub-G1 populations and cleavage of procaspase-3 and PARP, which could be prevented by pretreatment with the pan-caspase inhibitor, Z-VAD. Ectopic expression of miRNA-708 also increased the sensitivity to various apoptotic stimuli such as tumor necrosis factor-related apoptosis-inducing ligand, doxorubicin (Dox), and thapsigargin in Caki cells. Interestingly, miRNA-708 specifically repressed c-FLIPL without any change in c-FLIPs expression. In contrast, inhibition of endogenous miRNA-708 using antago-miRNAs resulted in an increase in c-FLIPL protein expression. The expression of c-FLIPL was upregulated in renal cell carcinoma (RCC) tissues compared to normal tissues. In contrast, miRNA-708 expression was reduced in RCC tissues. Finally, miRNA-708 enhanced the tumor-suppressive effect of Dox in a xenograft model of human RCC. In conclusion, miRNA-708 acts as a tumor suppressor because it negatively regulates the anti-apoptotic protein c-FLIPL and regulates the sensitivity of renal cancer cells to various apoptotic stimuli.

Altered microRNA expression profile in hepatitis B virus-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers, accounting for about 600,000 cancer deaths worldwide. Despite aggressive chemotherapy, the 5-year survival rate is less than 30% in the United States. This underscores the need for a better understanding of the molecular and cellular disease features. Many studies have demonstrated that aberrant regulation of microRNA (miRNA) expression plays a critical role in the development of various types of cancers including HCC. Here we analyzed the miRNA expression profile of HCC cases associated with chronic hepatitis B virus infection, one of the major etiologies of HCC. Our study identified 267 miRNAs that were differentially regulated in HCC specimens compared to adjacent normal tissues. We next analyzed putative target genes and the relevant signaling pathways that are regulated by these miRNAs. Our findings support the notion that dysfunction of miRNAs is linked to HCC pathogenesis and may lead to the identification of novel targets for diagnosing and treating HCC.

MicroRNA-124 regulates glucocorticoid sensitivity by targeting phosphodiesterase 4B in diffuse large B cell lymphoma.

Glucocorticoids (GCs) are chemotherapeutic drugs commonly used to treat hematological malignancies. However, a significant fraction of patients develop resistance to GCs during treatment. A better insight into how GC resistance develops is therefore needed. It was previously shown that cyclic AMP (cAMP) induces sensitivity to GCs by inhibiting the AKT/mTOR/MCL1 signaling, while high levels of phosphodiesterase 4B (PDE4B) reverse the effect of cAMP on GC responses in B-cell lymphoma. Here, we show that miR-124 influences GC-induced apoptosis by directly targeting PDE4B. Stable expression of miR-124 in diffuse large B cell lymphoma (DLBCL) cell lines diminished PDE4B expression. This was associated with increased cAMP levels, inhibition of the AKT/mTOR/MCL1 survival pathway, upregulation of GRα expression, and improved sensitivity to GCs in the presence of forskolin, an activator of adenylyl cyclase. Interestingly, miR-124 did not affect GC sensitivity in the absence of forskolin, indicating that the effect of this miRNA is accomplished via downregulation of PDE4B expression. Further, restoration of PDE4B expression in miR-124 cells rescued the phenotypic effect of this miRNA, demonstrating the critical role of PDE4B in miR-124-mediated regulation of the GC response. Our study supports the notion that miR-124 could be an attractive therapeutic target for overcoming GC resistance in DLBCL.