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Although inhibitors of epigenetic regulators have been effective in the treatment of cutaneous T cell lymphoma (CTCL) and other hematopoietic malignancies, they have been less effective in solid tumors, including ovarian cancer (OC). We have previously shown that inhibition of histone deacetylase (HDAC) activity induces expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (CXCL8, IL-8) in OC cells, resulting in their increased survival and proliferation. Here, we show that in addition to ovarian cancer SKOV3, OVCAR3, and CAOV3 cells, HDAC inhibition induces the CXCL8 expression in HeLa cells, but not in CTCL Hut-78 cells. In OC cells, the CXCL8 expression is induced by pharmacological inhibition of class I HDACs. Interestingly, while an individual suppression of HDAC1, HDAC2, or HDAC3 by corresponding siRNAs inhibits the CXCL8 expression, their simultaneous suppression induces the CXCL8 expression. The induced CXCL8 expression in OC cells is dependent on histone acetyltransferase (HAT) activity of CREB-binding protein (CBP), but not p300, and is associated with HAT-dependent p65 recruitment to CXCL8 promoter. Together, our results show that the CXCL8 expression in OC cells is induced by combined inhibition of HDAC1, -2, and -3, and silenced by suppression of HAT activity of CBP. In addition, our data indicate that the induced CXCL8 expression may be responsible for the limited effectiveness of HDAC inhibitors in OC and perhaps other solid cancers characterized by CXCL8 overexpression, and suggest that targeting class I HDACs and CBP may provide novel combination strategies by limiting the induced CXCL8 expression.

Pancreatic ductal adenocarcinoma (PDAC) is responsible for 7.3% of all cancer deaths. Even though there is a steady increase in patient survival for most cancers over the decades, the patient survival rate for pancreatic cancer remains low with current therapeutic strategies. The Wnt/β-catenin pathway controls the maintenance of somatic stem cells in many tissues and organs and is implicated in pancreatic carcinogenesis by regulating cell cycle progression, apoptosis, epithelial-mesenchymal transition (EMT), angiogenesis, stemness, tumor immune microenvironment, etc. Further, dysregulated Wnt has been shown to cause drug resistance in pancreatic cancer. Although different Wnt antagonists are effective in pancreatic patients, limitations remain that must be overcome to increase the survival benefits associated with this emerging therapy. In this review, we have summarized the role of Wnt signaling in pancreatic cancer and suggested future directions to enhance the survival of pancreatic cancer patients.

Ovarian cancer is associated with increased expression of the pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which induces tumor cell proliferation, angiogenesis, and metastasis. Even though bortezomib (BZ) has shown remarkable anti-tumor activity in hematological malignancies, it has been less effective in ovarian cancer; however, the mechanisms are not understood. We have recently shown that BZ unexpectedly induces the expression of IL-8 in ovarian cancer cells in vitro, by IκB kinase (IKK)-dependent mechanism. Here, we tested the hypothesis that IKK inhibition reduces the IL-8 production and increases BZ effectiveness in reducing ovarian tumor growth in vivo. Our results demonstrate that the combination of BZ and the IKK inhibitor Bay 117085 significantly reduces the growth of ovarian tumor xenografts in nude mice when compared to either drug alone. Mice treated with the BZ/Bay 117085 combination exhibit smallest tumors, and lowest levels of IL-8. Furthermore, the reduced tumor growth in the combination group is associated with decreased tumor levels of S536P-p65 NFκB and its decreased recruitment to IL-8 promoter in tumor tissues. These data provide the first in vivo evidence that combining BZ with IKK inhibitor is effective, and suggest that using IKK inhibitors may increase BZ effectiveness in ovarian cancer treatment.

Proinflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) contributes to ovarian cancer progression through its induction of tumor cell proliferation, survival, angiogenesis, and metastasis. Proteasome inhibition by bortezomib, which has been used as a frontline therapy in multiple myeloma, has shown only limited effectiveness in ovarian cancer and other solid tumors. However, the responsible mechanisms remain elusive. Here, we show that proteasome inhibition dramatically increases the IL-8 expression and release in ovarian cancer cells. The responsible mechanism involves an increased nuclear accumulation of IκB kinase β (IKKβ) and an increased recruitment of the nuclear IKKβ, p65-phosphorylated at Ser-536, and the transcription factor early growth response-1 (EGR-1) to the endogenous IL-8 promoter. Coimmunoprecipitation studies identified the nuclear EGR-1 associated with IKKβ and with p65, with preferential binding to S536P-p65. Both IKKβ activity and EGR-1 expression are required for the increased IL-8 expression induced by proteasome inhibition in ovarian cancer cells. Interestingly, in multiple myeloma cells the IL-8 release is not increased by bortezomib. Together, these data indicate that the increased IL-8 release may represent one of the underlying mechanisms responsible for the decreased effectiveness of proteasome inhibition in ovarian cancer treatment and identify IKKβ and EGR-1 as potential new targets in ovarian cancer combination therapies.

Overexpression of the pro-angiogenic chemokine IL-8 (CXCL8) is associated with a poor prognosis in several solid tumors, including epithelial ovarian cancer (EOC). Even though histone deacetylase (HDAC) inhibition has shown remarkable antitumor activity in hematological malignancies, it has been less effective in solid tumors, including EOC. Here we report results that may explain the decreased efficiency of HDAC inhibition in EOC, based on our data demonstrating that HDAC inhibition specifically induces expression of IL-8/CXCL8 in SKOV3, CAOV3, and OVCAR3 cells. Suppression or neutralization of vorinostat-induced IL-8/CXCL8 potentiates the vorinostat inhibitory effect on cell viability and proliferation. The IL-8/CXCL8 expression induced by vorinostat in EOC cells is dependent on IκB kinase (IKK) activity and associated with a gene-specific recruitment of IKKβ and IKK-dependent recruitment of p65 NFκB to the IL-8/CXCL8 promoter. In addition, HDAC inhibition induces acetylation of p65 and histone H3 and their IL-8/CXCL8 promoter occupancy. In vivo results demonstrate that combining vorinostat and the IKK inhibitor Bay 117085 significantly reduces tumor growth in nude mice compared with control untreated mice or either drug alone. Mice in the combination group had the lowest IL-8/CXCL8 tumor levels and the lowest tumor expression of the murine neutrophil [7/4] antigen, indicating reduced neutrophil infiltration. Together, our results demonstrate that HDAC inhibition specifically induces IL-8/CXCL8 expression in EOC cells and that the mechanism involves IKK, suggesting that using IKK inhibitors may increase the effectiveness of HDAC inhibitors when treating ovarian cancer and other solid tumors characterized by increased IL-8/CXCL8 expression.

AMP-activated protein kinase (AMPK) is an energy sensor and master regulator of metabolism. AMPK functions as a fuel gauge monitoring systemic and cellular energy status. Activation of AMPK occurs when the intracellular AMP/ATP ratio increases and leads to a metabolic switch from anabolism to catabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC), which catalyzes carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in de novo synthesis of fatty acids. AMPK thus regulates homeostasis of acetyl-CoA, a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Nucleocytosolic concentration of acetyl-CoA affects histone acetylation and links metabolism and chromatin structure. Here we show that activation of AMPK with the widely used antidiabetic drug metformin or with the AMP mimetic 5-aminoimidazole-4-carboxamide ribonucleotide increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation and altered gene expression in prostate and ovarian cancer cells. Direct inhibition of ACC with allosteric inhibitor 5-(tetradecyloxy)-2-furoic acid also increases acetylation of histones and non-histone proteins. Because AMPK activation requires liver kinase B1, metformin does not induce protein acetylation in liver kinase B1-deficient cells. Together, our data indicate that AMPK regulates the availability of nucleocytosolic acetyl-CoA for protein acetylation and that AMPK activators, such as metformin, have the capacity to increase protein acetylation and alter patterns of gene expression, further expanding the plethora of metformin's physiological effects.

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) counteract with each other to regulate gene expression by altering chromatin structure. Aberrant HDAC activity was reported in many human diseases including wide range of cancers, viral infections, cardiovascular complications, auto-immune diseases and kidney diseases. HDAC inhibitors are small molecules designed to block the malignant activity of HDACs. Chemokines and cytokines control inflammation, immunological and other key biological processes and are shown to be involved in various malignancies. Various HDACs and HDAC inhibitors were reported to regulate chemokines and cytokines. Even though HDAC inhibitors have remarkable anti-tumor activity in hematological cancers, they are not effective in treating many diseases and many patients relapse after treatment. However, the role of HDACs and cytokines in regulating these diseases still remain unclear. Therefore, understanding exact mechanisms and effector functions of HDACs are urgently needed to selectively inhibit them and to establish better a platform to combat various malignancies. In this review, we address regulation of chemokines and cytokines by HDACs and HDAC inhibitors and update on HDAC inhibitors in human diseases.

Expression of the proinflammatory and proangiogenic chemokine IL-8, which is regulated at the transcriptional level by NF-κB, is constitutively increased in androgen-independent metastatic prostate cancer and correlates with poor prognosis. Inhibition of NF-κB–dependent transcription was used as an anticancer strategy for the development of the first clinically approved 26S proteasome inhibitor, bortezomib (BZ). Even though BZ has shown remarkable antitumor activity in hematological malignancies, it has been less effective in prostate cancer and other solid tumors; however, the mechanisms have not been fully understood. In this article, we report that proteasome inhibition by BZ unexpectedly increases IL-8 expression in androgen-independent prostate cancer PC3 and DU145 cells, whereas expression of other NF-κB–regulated genes is inhibited or unchanged. The BZ-increased IL-8 expression is associated with increased in vitro p65 NF-κB DNA binding activity and p65 recruitment to the endogenous IL-8 promoter. In addition, proteasome inhibition induces a nuclear accumulation of IκB kinase (IKK)α, and inhibition of IKKα enzymatic activity significantly attenuates the BZ-induced p65 recruitment to IL-8 promoter and IL-8 expression, demonstrating that the induced IL-8 expression is mediated, at least partly, by IKKα. Together, these data provide the first evidence, to our knowledge, for the gene-specific increase of IL-8 expression by proteasome inhibition in prostate cancer cells and suggest that targeting both IKKα and the proteasome may increase BZ effectiveness in treatment of androgen-independent prostate cancer.

Interleukin-8 (IL-8), originally discovered as the neutrophil chemoattractant and inducer of leukocyte-mediated inflammation, contributes to cancer progression through its induction of tumor cell proliferation, survival, and migration. IL-8 expression is increased in many types of advanced cancers, including ovarian cancer, and correlates with poor prognosis. Bortezomib (BZ) is the first FDA-approved proteasome inhibitor that has shown remarkable antitumor activity in multiple myeloma and other hematological malignancies. In solid tumors, including ovarian carcinoma, BZ has been less effective as a single agent; however, the mechanisms remain unknown. We have recently shown that in ovarian cancer cells, BZ greatly increases IL-8 expression, while expression of other NFκB-regulated cytokines, IL-6 and TNF, is unchanged. In this chapter, we describe a protocol that uses real-time qRT-PCR to quantitatively analyze mRNA levels of IL-8 and IL-6 in BZ-treated ovarian cancer cells. The protocol can be easily modified and used for analysis of other cytokines in different cell types.

Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types.

Proteasome inhibition is a promising therapeutic strategy in a number of human cancers, including ovarian cancer, one of the most lethal human cancers. Bortezomib (BZ) is the first FDA approved pro...

Interleukin-8 (IL-8), originally discovered as the neutrophil chemoattractant and inducer of leukocyte-mediated inflammation, contributes to cancer progression through its induction of tumor cell proliferation, survival, and migration. IL-8 expression is increased in many types of advanced cancers, including ovarian cancer, and correlates with poor prognosis. Bortezomib (BZ) is the first FDA-approved proteasome inhibitor that has shown remarkable antitumor activity in multiple myeloma and other hematological malignancies. In solid tumors, including ovarian carcinoma, BZ has been less effective as a single agent; however, the mechanisms remain unknown. We have recently shown that in ovarian cancer cells, BZ greatly increases IL-8 expression, while expression of other NFκB-regulated cytokines, IL-6 and TNF, is unchanged. In this chapter, we describe a protocol that uses real-time qRT-PCR to quantitatively analyze mRNA levels of IL-8 and IL-6 in BZ-treated ovarian cancer cells. The protocol can be easily modified and used for analysis of other cytokines in different cell types.

Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types.