p38 Molecular Targeting for Next-Generation Multiple Myeloma Therapy.

Resistance to therapy and disease progression are the main causes of mortality in most cancers. In particular, the development of resistance is an important limitation affecting the efficacy of therapeutic alternatives for cancer, including chemotherapy, radiotherapy, and immunotherapy. Signaling pathways are largely responsible for the mechanisms of resistance to cancer treatment and progression, and multiple myeloma is no exception. p38 mitogen-activated protein kinase (p38) is downstream of several signaling pathways specific to treatment resistance and progression. Therefore, in recent years, developing therapeutic alternatives directed at p38 has been of great interest, in order to reverse chemotherapy resistance and prevent progression. In this review, we discuss recent findings on the role of p38, including recent advances in our understanding of its expression and activity as well as its isoforms, and its possible clinical role based on the mechanisms of resistance and progression in multiple myeloma.

Role of MicroRNA-7 (MiR-7) in Cancer Physiopathology.

miRNAs are non-coding RNA sequences of approximately 22 nucleotides that interact with genes by inhibiting their translation through binding to their 3' or 5' UTR regions. Following their discovery, the role they play in the development of various pathologies, particularly cancer, has been studied. In this context, miR-7 is described as an important factor in the development of cancer because of its role as a tumor suppressor, regulating a large number of genes involved in the development and progression of cancer. Recent data support the function of miR-7 as a prognostic biomarker in cancer, and miR-7 has been proposed as a strategy in cancer therapy. In this work, the role of miR-7 in various types of cancer is reviewed, illustrating its regulation, direct targets, and effects, as well as its possible relationship to the clinical outcome of cancer patients.

Roles and Regulation of BCL-xL in Hematological Malignancies.

Members of the Bcl-2 family are proteins that play an essential role in the regulation of apoptosis, a crucial process in development and normal physiology in multicellular organisms. The essential mechanism of this family of proteins is given by the role of pro-survival proteins, which inhibit apoptosis by their direct binding with their counterpart, the effector proteins of apoptosis. This family of proteins was named after the typical member Bcl-2, which was named for its discovery and abnormal expression in B-cell lymphomas. Subsequently, the structure of one of its members BCL-xL was described, which allowed one to understand much of the molecular mechanism of this family. Due to its role of BCL-xL in the regulation of cell survival and proliferation, it has been of great interest in its study. Due to this, it is important to research its role regarding the development and progression of human malignancies, especially in hematologic malignancies. Due to its variation in expression in cancer, it has been suggested that BCL-xL can or cannot play a role in cancer depending on the cellular or tissue context. This review discusses recent advances in its transcriptional regulation of BCL-xL, as well as the advances regarding the activities of BCL-xL in hematological malignancies, its possible role as a biomarker, and its possible clinical relevance in these malignancies.

KLF4 inhibition by Kenpaullone induces cytotoxicity and chemo sensitization in B-NHL cell lines via YY1 independent.

Krüppel-like factor 4 (KLF4) is a member of the KLF transcription factor family containing zinc-fingers, and is involved in the regulation of apoptosis, proliferation and differentiation of B cells and B-cell malignancies. KLF4 can act like an oncogene, we shown that KLF4 overexpression correlated with poor prognostic and chemoresistance in B-NHL. In addition, we shown that KLF4 is regulated by YY1. In this study, we demonstrate that chemical inhibition of KLF4 by Kenpaullone, results in suppression of proliferation, cell survival, downregulation of Bcl-2 and increases apoptosis in B-NHL cell lines through YY1 independent pathway. Combination of Kenpaullone and Doxorubicin, increased apoptosis. The co-expressions of KLF4/YY1 or KLF4/Bcl-2 in NHL was analyzed using Oncomine Database, exhibiting a positive correlation of expression. The present findings suggest that the chemical inhibition of KLF4 by Kenpaullone treatment could be a potential therapeutic alternatively in KLF4+ lymphomas.

Function of Deptor and its roles in hematological malignancies.

Deptor is a protein that interacts with mTOR and that belongs to the mTORC1 and mTORC2 complexes. Deptor is capable of inhibiting the kinase activity of mTOR. It is well known that the mTOR pathway is involved in various signaling pathways that are involved with various biological processes such as cell growth, apoptosis, autophagy, and the ER stress response. Therefore, Deptor, being a natural inhibitor of mTOR, has become very important in its study. Because of this, it is important to research its role regarding the development and progression of human malignancies, especially in hematologic malignancies. Due to its variation in expression in cancer, it has been suggested that Deptor can act as an oncogene or tumor suppressor depending on the cellular or tissue context. This review discusses recent advances in its transcriptional and post-transcriptional regulation of Deptor. As well as the advances regarding the activities of Deptor in hematological malignancies, its possible role as a biomarker, and its possible clinical relevance in these malignancies.

MicroRNA-7 Regulates Migration and Chemoresistance in Non-Hodgkin Lymphoma Cells Through Regulation of KLF4 and YY1.

The discovery and description of the role of microRNAs has become very important, specifically due to their participation in the regulation of proteins and transcription factors involved in the development of cancer. microRNA-7 (miR-7) has been described as a negative regulator of several proteins involved in cancer, such as YY1 and KLF4. We have recently reported that YY1 and KLF4 play a role in non-Hodgkin lymphoma (NHL) and that the expression of KLF4 is regulated by YY1. Therefore, in this study we analyzed the role of miR-7 in NHL through the negative regulation of YY1 and KLF4. qRT-PCR showed that there is an inverse expression of miR-7 in relation to the expression of YY1 and KLF4 in B-NHL cell lines. The possible regulation of YY1 and KLF4 by miR-7 was analyzed using the constitutive expression or inhibition of miR-7, as well as using reporter plasmids containing the 3 'UTR region of YY1 or KLF4. The role of miR-7 in NHL, through the negative regulation of YY1 and KLF4 was determined by chemoresistance and migration assays. We corroborated our results in cell lines, in a TMA from NHL patients including DLBCL and follicular lymphoma subtypes, in where we analyzed miR-7 by ISH and YY1 and KLF4 using IHC. All tumors expressing miR-7 showed a negative correlation with YY1 and KLF4 expression. In addition, expression of miR-7 was analyzed using the GEO Database; miR-7 downregulated expression was associated with pour overall-survival. Our results show for the first time that miR-7 is implicate in the cell migration and chemoresistance in NHL, through the negative regulation of YY1 and KLF4. That also support the evidence that YY1 and KLF4 can be a potential therapeutic target in NHL.

A Novel Therapeutic Induces DEPTOR Degradation in Multiple Myeloma Cells with Resulting Tumor Cytotoxicity.

Prior work indicates DEPTOR expression in multiple myeloma cells could be a therapeutic target. DEPTOR binds to mTOR via its PDZ domain and inhibits mTOR kinase activity. We previously identified a drug, which prevented mTOR-DEPTOR binding (NSC126405) and induced multiple myeloma cytotoxicity. We now report on a related therapeutic, drug 3g, which induces proteasomal degradation of DEPTOR. DEPTOR degradation followed drug 3g binding to its PDZ domain and was not due to caspase activation or enhanced mTOR phosphorylation of DEPTOR. Drug 3g enhanced mTOR activity, and engaged the IRS-1/PI3K/AKT feedback loop with reduced phosphorylation of AKT on T308. Activation of TORC1, in part, mediated multiple myeloma cytotoxicity. Drug 3g was more effective than NSC126405 in preventing binding of recombinant DEPTOR to mTOR, preventing binding of DEPTOR to mTOR inside multiple myeloma cells, in activating mTOR and inducing apoptosis in multiple myeloma cells. In vivo, drug 3g injected daily abrogated DEPTOR expression in xenograft tumors and induced an antitumor effect although modest weight loss was seen. Every-other-day treatment, however, was equally effective without weight loss. Drug 3g also reduced DEPTOR expression in normal tissues. Although no potential toxicity was identified in hematopoietic or hepatic function, moderate cardiac enlargement and glomerular mesangial hypertrophy was seen. DEPTOR protected multiple myeloma cells against bortezomib suggesting anti-DEPTOR drugs could synergize with proteasome inhibitors (PI). Indeed, combinations of drug NSC126405 + bortezomib were synergistic. In contrast, drug 3g was not and was even antagonistic. This antagonism was probably due to prevention of proteasomal DEPTOR degradation.

Regulation of Krüppel-Like Factor 4 (KLF4) expression through the transcription factor Yin-Yang 1 (YY1) in non-Hodgkin B-cell lymphoma.

Krüppel-Like Factor 4 (KLF4) is a member of the KLF transcription factor family, and evidence suggests that KLF4 is either an oncogene or a tumor suppressor. The regulatory mechanism underlying KLF4 expression in cancer, and specifically in lymphoma, is still not understood. Bioinformatics analysis revealed two YY1 putative binding sites in the KLF4 promoter region (-950 bp and -105 bp). Here, the potential regulation of KLF4 by YY1 in NHL was analyzed. Mutation of the putative YY1 binding sites in a previously reported system containing the KLF4 promoter region and CHIP analysis confirmed that these binding sites are important for KLF4 regulation. B-NHL cell lines showed that both KLF4 and YY1 are co-expressed, and transfection with siRNA-YY1 resulted in significant inhibition of KLF4. The clinical implications of YY1 in the transcriptional regulation of KLF4 were investigated by IHC in a TMA with 43 samples of subtypes DLBCL and FL, and all tumor tissues expressing YY1 demonstrated a correlation with KLF4 expression, which was consistent with bioinformatics analyses in several databases. Our findings demonstrated that KLF4 can be transcriptionally regulated by YY1 in B-NHL, and a correlation between YY1 expression and KLF4 was found in clinical samples. Hence, both YY1 and KLF4 may be possible therapeutic biomarkers of NHL.

Cytotoxic effect caspase activation dependent of a genetically engineered fusion protein with a CD154 peptide mimetic (OmpC-CD154p) on B-NHL cell lines is mediated by the inhibition of bcl-6 and YY1 through MAPK p38 activation.

The interaction between CD40, and its ligand, CD154, is essential for the development of humoral and cellular immune responses. The selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically based diseases and malignancies. We are developing a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Tyr140-Ser-149 amino acid strand. This OmpC-CD154 binds CD40 and activates B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth, proliferation and induced apoptosis in the B-NHL cell lines Raji and Ramos. The Bcl-2 family proteins were regulated and the Bcl-6 and YY1 oncoproteins were inhibited. p38 MAPK activation is an important mechanism underlying the effect on proliferation and apoptosis mediated by this fusion protein. This study establishes a basis for the possible use of fusion protein OmpC-CD154 as an alternative treatment for B-NHL.

Dual role of hypoxia-inducible factor 1 α in experimental pulmonary tuberculosis: its implication as a new therapeutic target.

AIM: Investigate the role of hypoxia-inducible factor-1α (HIF-1α) in pulmonary tuberculosis (TB). METHODS & RESULTS: A model of progressive pulmonary TB in BALB/c mice, immunohistochemistry and digital pathology were used. High HIF-1α expression was observed during early TB in activated macrophages. During late TB, even higher HIF-1α expression was observed in foamy macrophages, which are resistant to apoptosis. Blocking HIF-1α during early infection with 2-methoxyestradiol worsened the disease, while during late TB, it induced macrophage apoptosis and decreased bacillary loads. CONCLUSION: HIF-1α has a dual role in experimental TB. This finding could have therapeutic implications because combined treatment with 2-methoxyestradiol and antibiotics appeared to eliminate mycobacteria more efficiently than conventional chemotherapy during advanced disease.

Role of Yin Yang-1 (YY1) in the transcription regulation of the multi-drug resistance (MDR1) gene.

Resistance to chemotherapy hinders the successful treatment of acute lymphoblastic leukemia (ALL). The multi-drug resistance-1 (MDR1/ABCB1) gene encodes P-glycoprotein (P-gp), which plays an important role in chemoresistance; however, its transcriptional regulation remains unclear. We investigated the role of YY1 in the regulation of MDR1 and its relation to ALL outcomes. Analysis of the MDR1 promoter revealed four putative YY1-binding sites, which we analyzed using a reporter system and ChIP analysis. YY1 silencing resulted in the inhibition of MDR1 expression and function. The clinical roles of YY1 and MDR1 expression were evaluated in children with ALL. Expression of both proteins was increased in ALL patients compared to controls. We identified a positive correlation between YY1 and MDR1 expression. High levels of YY1 were associated with decreased overall survival. Our results demonstrated that YY1 regulates the transcription of MDR1. Therefore, YY1 may serve as a useful prognostic and/or therapeutic target.

[Corrigendum] Death receptor 5 expression is inversely correlated with prostate cancer progression.

During the preparation of the figures in the above article, the authors inadvertently duplicated in Fig. 1B, a and b (high and low magnification images) the images that had already appeared as Figs. 5A, a and c (high and low magnification images), respectively, of the following paper: Huerta-Yepez S, Baritaki S, Baay-Guzman G, Hernandez-Luna MA, Hernandez-Cueto A, Vega MI and Bonavida B: Contribution of either YY1 or BclXL-induced inhibition by the NO-donor DETANONOate in the reversal of drug resistance, both in vitro and in vivo. Nitric Oxide 29: 17-24, 2013. The revised version of Fig. 1 containing the corrected data for Fig. 1B, a and b (high and low magnification images; the YY1 data) is shown opposite protein expression. All those authors whom the corresponding author was able to contact have agreed to this Corrigendum. The authors regret this error, and apologize for any confusion that it may have caused. [the original article was published in the Molecular Medicine Reports 10: 2279-2286, 2014; DOI: 10.3892/mmr.2014.2504 ].

Sulforaphane induces differential modulation of mitochondrial biogenesis and dynamics in normal cells and tumor cells.

Antioxidant-based chemotherapy has been intensely debated. Herein, we show that sulforaphane (SFN) induced mitochondrial biogenesis followed by mitochondrial fusion in a kidney cell line commonly used in nephroprotective models. At the same concentration and exposure time, SFN induced cell death in prostate cancer cells accompanied by mitochondrial biogenesis and fragmentation. Stabilization of the nuclear factor E2-related factor-2 (Nrf2) could be associated with these effects in the tumor cell line. An increase in the peroxisome proliferator-activated receptor-γ co-activator-1α (PGC1α) level and a decrease in the hypoxia-inducible factor-1α (HIF1α) level would suggest a possible metabolic shift. The knockdown in the nuclear respiratory factor-1 (NRF1) attenuated the SFN-induced effect on prostate cancer cells demonstrating that mitochondrial biogenesis plays an important role in cell death for this kind of tumor cells. This evidence supports SFN as a potential antineoplastic agent that could inhibit tumor development and could protect normal tissues by modulating common processes.

A novel role of Yin-Yang-1 in pulmonary tuberculosis through the regulation of the chemokine CCL4.

Mycobacterium tuberculosis (M. tb) is the etiological agent of pulmonary tuberculosis (TB); this disease remains a worldwide health problem. Yin-Yang-1 (YY1) plays a major role in the maintenance and progression of some pulmonary diseases, including pulmonary fibrosis. However, the role of YY1 in TB remains unknown. The aim of this study was to elucidate the role of YY1 in the regulation of CCL4 and its implication in TB. We determined whether YY1 regulates CCL4 using reporter plasmids, ChIP and siRNA assays. Immunohistochemistry and digital pathology were used to measure the expression of YY1 and CCL4 in a mouse model of TB. A retrospective comparison of patients with TB and control subjects was used to measure the expression of YY1 and CCL4 using tissue microarrays. Our results showed that YY1 regulates the transcription of CCL4; moreover, YY1, CCL4 and TGF-ß were overexpressed in the lung tissues of mice with TB during the late stages of the disease and the tissues of TB patients. The expression of CCL4 and TGF-ß correlated with YY1 expression. In conclusion, YY1 regulates CCL4 transcription; moreover, YY1 is overexpressed in experimental and human TB and is positively correlated with CCL4 and TGF-ß expression. Therefore, treatments that decrease YY1 expression may be a new therapeutic strategy against TB.

P38 MAPK expression and activation predicts failure of response to CHOP in patients with Diffuse Large B-Cell Lymphoma.

BACKGROUND: The p38 MAPK is constitutively activated in B-NHL cell lines and regulates chemoresistance. Accordingly, we hypothesized that activated p38 MAPK may be associated with the in vivo unresponsiveness to chemotherapy in B-NHL patients. METHODS: Tissue microarrays generated from eighty untreated patients with Diffused Large B Cell Lymphoma (DLBCL) were examined by immunohistochemistry for the expression of p38 and phospho p38 (p-p38) MAPK. In addition, both Bcl-2 and NF-κB expressions were determined. Kaplan Meier analysis was assessed. RESULTS: Tumor tissues expressed p38 MAPK (82 %) and p-p38 MAPK (30 %). Both p38 and p-p38 MAPK expressions correlated with the high score performance status. A significant correlation was found between the expression p-p38 and poor response to CHOP. The five year median follow-up FFS was 81 % for p38(-) and 34 % for p38(+) and for OS was 83 % for p38(-) and 47 % for p38(+). The p-p38(+) tissues expressed Bcl-2 and 90 % of p-p38(-) where Bcl-2(-). The coexpression of p-p38 and Bcl-2 correlated with pool EFS and OS. There was no correlation between the expression of p-p38 and the expression of NF-κB. CONCLUSION: The findings revealed, for the first time, that a subset of patients with DLBCL and whose tumors expressed high p-p38 MAPK responded poorly to CHOP therapy and had poor EFS and OS. The expression of p38, p-p38, Bcl2 and the ABC subtype are significant risk factors both p38 and p-p38 expressions remain independent prognostic factors.

Overcoming rituximab drug-resistance by the genetically engineered anti-CD20-hIFN-α fusion protein: Direct cytotoxicity and synergy with chemotherapy.

Treatment of patients with B-NHL with rituximab and CHOP has resulted in significant clinical responses. However, a subset of patients develops resistance to further treatments. The mechanism of unresponsiveness in vivo is not known. We have reported the development of rituximab-resistant clones derived from B-NHL cell lines as models to investigate the mechanism of resistance. The resistant clones exhibit hyper-activated survival/anti-apoptotic pathways and no longer respond to a combination of rituximab and drugs. Recent studies reported the therapeutic efficacy in mice bearing B-cell lymphoma xenografts following treatment with the anti-CD20-hIFNα fusion protein. We hypothesized that the fusion protein may bypass rituximab resistance and inhibit survival signaling pathways. Treatment of the rituximab-resistant clones with anti-CD20-hIFNα, but not with rituximab, IFNα, or rituximab+IFNα resulted in significant inhibition of cell proliferation and induction of cell death. Treatment with anti-CD20-hIFNα sensitized the cells to apoptosis by CDDP, doxorubicin and Treanda. Treatment with anti-CD20-hIFNα inhibited the NF-κB and p38 MAPK activities and induced the activation of PKC-δ and Stat-1. These effects were corroborated by the use of the inhibitors SB203580 (p38 MAPK) and Rottlerin (PKC-δ). Treatment with SB203580 enhanced the sensitization of the resistant clone by anti-CD20-hIFNα to CDDP apoptosis. In contrast, treatment with Rotterin inhibited significantly the sensitization induced by anti-CD20-hIFNα. Overall, the findings demonstrate that treatment with anti-CD20-hIFNα reverses resistance of B-NHL. These findings suggest the potential application of anti-CD20-hIFNα in combination with drugs in patients unresponsive to rituximab-containing regimens.

Inhibition of tumor progression during allergic airway inflammation in a murine model: significant role of TGF-ß.

INTRODUCTION: TGF-ß is an important mediator of pulmonary allergic inflammation, and it has been recently reported to be a potential inhibitor of lung tumor progression. The correlation between cancer and allergic inflammatory diseases remains controversial. Thus, the aim of the present study was to evaluate the effects of pulmonary allergic inflammation and in particular the role of TGF-ß on cancer progression. METHODS: Cancer cells were implanted in a BALB/c mice model of allergic airway inflammation, and tumor growth was measured. Apoptosis was evaluated by TUNEL assay, and TGF-ß was measured by ELISA. Expression of proliferating cell nuclear antigen, TGF-ß, TGF-ß receptors I and II, phospho-Smad2 and phospho-Smad4 was evaluated by immunohistochemistry and quantified using digital pathology. The effect of a TGF-ß activity inhibitor and recombinant TGF-ß on tumor growth was analyzed. The effect of exogenous TGF-ß on cell proliferation and apoptosis was evaluated in vitro. RESULTS: Mice with allergic airway inflammation exhibited decreased tumor volumes due to cell proliferation inhibition and increased apoptosis. TGF-ß was increased in the sera and tumor tissues of allergic mice. TGF-ß activity inhibition increased tumor progression in allergic mice by enhancing proliferation and decreasing apoptosis of tumor cells. The administration of TGF-ß resulted in reduced tumor growth. CONCLUSION: This study is the first to establish an inverse relationship between allergic airway inflammation and tumor progression. This effect appears to be mediated by TGF-ß, which is overexpressed in tumor cells during pulmonary allergic inflammation. This study indicates that TGF-ß is a potential target for antitumor therapy.

Death receptor 5 expression is inversely correlated with prostate cancer progression.

Prostate carcinoma (PCa) is one of the most common cancers in men. Prostate-specific antigen (PSA) has been widely used to predict the outcome of PCa and screening with PSA has resulted in a decline in mortality. However, PSA is not an optimal prognostic tool as its sensitivity may be too low to reduce morbidity and mortality. Consequently, there is a demand for additional robust biomarkers for prostate cancer. Death receptor 5 (DR5) has been implicated in the prognosis of several cancers and it has been previously shown that it is negatively regulated by Yin Yang 1 (YY1) in prostate cancer cell lines. The present study investigated the clinical significance of DR5 expression in a prostate cancer patient cohort and its correlation with YY1 expression. Immunohistochemical analysis of protein expression distribution was performed using tissue microarray constructs from 54 primary PCa and 39 prostatic intraepithelial neoplasia (PIN) specimens. DR5 expression was dramatically reduced as a function of higher tumor grade. By contrast, YY1 expression was elevated in PCa tumors as compared with that in PIN, and was increased with higher tumor grade. DR5 had an inverse correlation with YY1 expression. Bioinformatic analyses corroborated these data. The present findings suggested that DR5 and YY1 expression levels may serve as progression biomarkers for prostate cancer.

Overexpression of Yin Yang 1 in bone marrow-derived human multiple myeloma and its clinical significance.

Multiple myeloma (MM) patients initially respond to conventional therapy, however, many develop resistance and have recurrences. We have reported in other tumors that the transcription factor Yin Yang 1 (YY1) is a resistant factor and, thus, we hypothesized that YY1 may be over-expressed in MM. Significantly, higher expression (staining intensity and cell frequency) of YY1 in MM cell lines and in bone marrow-derived (BM) MM from 22 MM patients was observed as compared to expression in normal BM. Higher nuclear YY1 staining was associated with disease progression. Bioinformatic analyses of mRNA in data sets corroborated the above findings and showed significant overexpression of YY1 in MM compared to normal tissues and other hematopoietic disorders. The role of YY1 expression in the regulation of drug resistance was exemplified in a drug-resistant MM cell line transfected with YY1 siRNA and which was shown to be sensitized to bortezomib-induced apoptosis. These findings highlight the potential prognostic significance of YY1 expression level in MM patients and as a therapeutic target.

Expression of KLF4 is a predictive marker for survival in pediatric Burkitt lymphoma.

Krüppel-like factor 4 (KLF4) is expressed in a variety of tissues with diverse physiological functions and activities. KLF4 can also function as a tumor suppressor or an oncogene, depending on the cellular context. Its role in hematological malignancies is controversial. This study examined the expression levels of KLF4 by immunohistochemistry in 73 pediatric non-Hodgkin lymphomas (NHLs) in a tissue microarray and also on several B-NHL cell lines. Elevated levels of KLF4 expression were detected in 66% of lymphoma cases and were more frequent in the Burkitt lymphoma (p = 0.05) subtype. There was a significant predictive power for outcome with low KLF4 expression, predicting a favorable overall survival compared to high levels. Multivariate analyses confirmed the association of KLF4 expression with unfavorable overall survival (p < 0.005). These findings were consistent with analyses in existing NHL microarray datasets. The present findings revealed that KLF4 is overexpressed in Burkitt pediatric lymphoma and is a potential biomarker for inferior overall survival.