16 research outputs found
Maspin differential expression patterns as a potential marker for targeted screening of esophageal adenocarcinoma/gastroesophageal junction adenocarcinoma.
Barrett's esophagus (BE) is a predisposing factor of esophageal adenocarcinoma/gastroesophageal junction adenocarcinoma (ECA/GEJ Aca). BE patients are stratified and subsequently monitored according to the risk of malignant progression by the combination of endoscopy and biopsy. This study is to evaluate the maspin expression patterns as early diagnostic markers of malignancy in BE patients. Immunohistochemistry (IHC) staining was performed on 62 archival core biopsies from 35 patients, including BE without dysplasia (intestinal metaplasia, IM), BE with low grade dysplasia, BE with high grade dysplasia, carcinoma in situ, and well to poorly differentiated ECA/GEJ Aca (PD-ECA/GEJ Aca). The intensity and the subcellular distribution of immunoreactivity were evaluated microscopically. Statistical analysis was performed using the χ2 and Fisher exact tests. The level of epithelial-specific tumor suppressor maspin protein inversely correlated with the progression from IM to PD-ECA/GEJ Aca. Lesions of each pathological grade could be divided into subtypes that exhibited distinct maspin subcellular distribution patterns, including nuclear only (Nuc), combined nuclear and cytoplasmic (Nuc+Cyt), cytoplasmic only (Cyt) and overall negligible (Neg). The Cyt subtype, which was minor in both IM and dysplasia (approximately 10%), was predominant in ECA/GEJ Aca as early as well-differentiated lesions (more than 50%: p = 0.0092). In comparison, nuclear staining of the tumor suppressor TP53 was heterogeneous in dysplasia, and did not correlate with the differentiation grades of ECA/GEJ Aca. The Cyt subtype of maspin expression pattern in core biopsies of BE patients may serve as a molecular marker for early diagnosis of ECA/GEJ Aca.This work was supported by the NIH grant P30CA022453 (to the Karmanos Cancer Institute with Sheng, S. as a program leader), the Ruth Sager Memorial Fund (to Sheng, S.), the Karmanos Cancer Institute Pilot Project Grant 25S5Z (to Sheng, S.), and the Karmanos Cancer Institute Prostate Cancer Research Pilot Project Grant (to Sheng, S.)
Tumor suppressor maspin as a modulator of host immune response to cancer
Despite the promising clinical outcome, the primary challenge of the curative cancer immunotherapy is to overcome the dichotomy of the immune response: tumor-evoked immunostimulatory versus tumor-induced immunosuppressive. The goal needs to be two-fold, to re-establish sustainable antitumor-cancer immunity and to eliminate immunosuppression. The successful elimination of cancer cells by immunosurveillance requires the antigenic presentation of the tumor cells or tumor-associated antigens and the expression of immunostimulatory cytokines and chemokines by cancer and immune cells. Tumors are heterogeneous and as such, some of the tumor cells are thought to have stem cell characteristics that enable them to suppress or desensitize the host immunity due to acquired epigenetic changes. A central mechanism underlying tumor epigenetic instability is the increased histone deacetylase (HDAC)-mediated repression of HDAC-target genes regulating homeostasis and differentiation. It was noted that pharmacological HDAC inhibitors are not effective in eliminating tumor cells partly because they may induce immunosuppression. We have shown that epithelial-specific tumor suppressor maspin, an ovalbumin-like non-inhibitory serine protease inhibitor, reprograms tumor cells toward better differentiated phenotypes by inhibiting HDAC1. Recently, we uncovered a novel function of maspin in directing host immunity towards tumor elimination. In this review, we discuss the maspin and maspin/HDAC1 interplay in tumor biology and immunology. We propose that maspin based therapies may eradicate cancer
Cotargeting of Mitochondrial Complex I and Bcl-2 Shows Antileukemic Activity against Acute Myeloid Leukemia Cells Reliant on Oxidative Phosphorylation
Targeting oxidative phosphorylation (OXPHOS) is a promising strategy to improve treatment outcomes of acute myeloid leukemia (AML) patients. IACS-010759 is a mitochondrial complex I inhibitor that has demonstrated preclinical antileukemic activity and is being tested in Phase I clinical trials. However, complex I deficiency has been reported to inhibit apoptotic cell death through prevention of cytochrome c release. Thus, combining IACS-010759 with a BH3 mimetic may overcome this mechanism of resistance leading to synergistic antileukemic activity against AML. In this study, we show that IACS-010759 and venetoclax synergistically induce apoptosis in OXPHOS-reliant AML cell lines and primary patient samples and cooperatively target leukemia progenitor cells. In a relatively OXPHOS-reliant AML cell line derived xenograft mouse model, IACS-010759 treatment significantly prolonged survival, which was further enhanced by treatment with IACS-010759 in combination with venetoclax. Consistent with our hypothesis, IACS-010759 treatment indeed retained cytochrome c in mitochondria, which was completely abolished by venetoclax, resulting in Bak/Bax- and caspase-dependent apoptosis. Our preclinical data provide a rationale for further development of the combination of IACS-010759 and venetoclax for the treatment of patients with AML
Identification of an Intrinsic Determinant Critical for Maspin Subcellular Localization and Function
<div><p>Maspin, a multifaceted tumor suppressor, belongs to the serine protease inhibitor superfamily, but only inhibits serine protease-like enzymes such as histone deacetylase 1 (HDAC1). Maspin is specifically expressed in epithelial cells and it is differentially regulated during tumor progression. A new emerging consensus suggests that a shift in maspin subcellular localization from the nucleus to the cytoplasm stratifies with poor cancer prognosis. In the current study, we employed a rational mutagenesis approach and showed that maspin reactive center loop (RCL) and its neighboring sequence are critical for maspin stability. Further, when expressed in multiple tumor cell lines, single point mutation of Aspartate<sup>346</sup> (D<sup>346</sup>) to Glutamate (E<sup>346</sup>), maspin<sup>D346E</sup>, was predominantly nuclear, whereas wild type maspin (maspin<sup>WT</sup>) was both cytoplasmic and nuclear. Evidence from cellular fractionation followed by immunological and proteomic protein identification, combined with the evidence from fluorescent imaging of endogenous proteins, fluorescent protein fusion constructs, as well as bimolecular fluorescence complementation (BiFC) showed that the increased nuclear enrichment of maspin<sup>D346E</sup> was, at least in part, due to its increased affinity to HDAC1. Maspin<sup>D346E</sup> was also more potent than maspin<sup>WT</sup> as an HDAC inhibitor. Taken together, our evidence demonstrates that D<sup>346</sup> is a critical <i>cis</i>-element in maspin sequence that determines the molecular context and subcellular localization of maspin. A mechanistic model derived from our evidence suggests a new window of opportunity for the development of maspin-based biologically competent HDAC inhibitors for cancer treatment.</p></div
Targeted therapy of pyrrolo[2,3-d]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment
Novel therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. In addition, therapies that target unique vulnerabilities in the tumor microenvironment (TME) of EOC have largely been unrealized. One strategy to achieve selective drug delivery for EOC therapy involves use of targeted antifolates via their uptake by folate receptor (FR) proteins, resulting in inhibition of essential one-carbon (C1) metabolic pathways. FRα is highly expressed in EOCs, along with the proton-coupled folate transporter (PCFT); FRβ is expressed on activated macrophages, a major infiltrating immune population in EOC. Thus, there is great potential for targeting both the tumor and the TME with agents delivered via selective transport by FRs and PCFT. In this report, we investigated the therapeutic potential of a novel cytosolic C1 6-substituted pyrrolo[2,3-d]pyrimidine inhibitor AGF94, with selectivity for uptake by FRs and PCFT and inhibition of de novo purine nucleotide biosynthesis, against a syngeneic model of ovarian cancer (BR-Luc) which recapitulates high-grade serous ovarian cancer in patients. In vitro activity of AGF94 was extended in vivo against orthotopic BR-Luc tumors. With late-stage subcutaneous BR-Luc xenografts, AGF94 treatment resulted in substantial anti-tumor efficacy, accompanied by significantly decreased M2-like FRβ-expressing macrophages and increased CD3+ T cells, whereas CD4+ and CD8+ T cells were unaffected. Our studies demonstrate potent anti-tumor efficacy of AGF94 in the therapy of EOC in the context of an intact immune system, and provide a framework for targeting the immunosuppressive TME as an essential component of therapy
Increased nuclear localization of maspin<sup>D346E</sup> correlates with increased HDAC1 interaction.
<p>(<b>A</b>) Confocal imaging of endogenous maspin (green) and HDAC1 (red) immunofluorescence staining in MCF10A cells. Scale bar = 20 µm (<b>B</b>). Bimolecular fluorescence complementation (BIFC) of live PC3 and DU145 cells transiently transected with pBiFC maspin<sup>WT</sup> YC or pBiFC maspin<sup>D346E</sup> YC in combination with pBiFC HDAC1 YN. The BiFC of bJunYN and BiFC bFosYC and BiFC of bJunYN and BiFC bFosYC Δ179–193 were used as positive and negative controls, respectfully, in PC3 cells. Scale bar = 10 µm (<b>C</b>) Immunofluorescence imaging (x40) of live PC3 cells after co-transfection with either pEGFP N1-maspin<sup>WT</sup> or pEGFP N1 maspin<sup>D346E</sup> in combination with pcDNA3.1 HDAC1-RFP. → indicates cytosolic maspin/HDAC1 interaction and ▸ indicates nuclear maspin/HDAC1 interaction. Scale bar = 20 µm. (<b>D</b>) Western blot of recombinant maspin and HDAC1 after immunoprecipitation (IP) with maspin antibody in DU145 cells. The mouse IgG was used as a negative control. Total levels of maspin, HDAC1, and loading control GAPDH are shown in the input panel. Numbers below represent normalized HDAC1/maspin ratio.</p
A hypothetical model explaining disregulation of maspin in tumor progression.
<p>(<b>A</b>) Benign tumor or better differentiated cancer with better prognosis is associated with nuclear maspin. Nuclear maspin has a stronger affinity towards HDAC1 as compared to hypothetical HDAC1-associated nuclear factor X. (<b>B</b>) High grade carcinoma exhibiting a less differentiated phenotype and worse prognosis is associated with cytosolic maspin or loss of maspin. In cancer, hypothetical HDAC1- associated nuclear factor X is X′ and it has a stronger affinity towards HDAC1 as compared to maspin. (<b>C</b>) Therapeutic potential of bioengineered maspin mutant or its derivative in treating advanced disease. Maspin mutant restores maspin nuclear localization and HDAC1 inhibition in advanced disease.</p
Maspin nuclear localization correlates with increased histone acetylation and release of HDAC-repressed gene expression.
<p>(<b>A</b>) Western blot of recombinant maspin, HDAC1, and HDAC1 target protein (acetylated Histone 3 at Lysine 9 (H3 Acetyl K<sup>9</sup>)) in DU145 cells. GAPDH was used as a loading control. (<b>B</b>) Q-RT-PCR of HDAC1 targeted genes differentially regulated by maspin. The threshold cycle (ct) numbers obtained from qRT-PCR were normalized by the internal GAPDH controls and presented as the fold change. Maspin<sup>WT</sup>: black bar; maspin<sup>D346E</sup>: white bar.</p
The combination of CUDC-907 and gilteritinib shows promising in vitro and in vivo antileukemic activity against FLT3-ITD AML
Abstract About 25% of patients with acute myeloid leukemia (AML) harbor FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations and their prognosis remains poor. Gilteritinib is a FLT3 inhibitor approved by the US FDA for use in adult FLT3-mutated relapsed or refractory AML patients. Monotherapy, while efficacious, shows short-lived responses, highlighting the need for combination therapies. Here we show that gilteritinib and CUDC-907, a dual inhibitor of PI3K and histone deacetylases, synergistically induce apoptosis in FLT3-ITD AML cell lines and primary patient samples and have striking in vivo efficacy. Upregulation of FLT3 and activation of ERK are mechanisms of resistance to gilteritinib, while activation of JAK2/STAT5 is a mechanism of resistance to CUDC-907. Gilteritinib and CUDC-907 reciprocally overcome these mechanisms of resistance. In addition, the combined treatment results in cooperative downregulation of cellular metabolites and persisting antileukemic effects. CUDC-907 plus gilteritinib shows synergistic antileukemic activity against FLT3-ITD AML in vitro and in vivo, demonstrating strong translational therapeutic potential