Molecular Target For Anticancer Therapy Research Articles

Structure Modeling of Human TyrosylDNA Phosphodiesterase 1 and Screening for Its Inhibitors

The DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1) represents a potential molecular target for anticancer therapy. A human Tdp1 model has been constructed using the methods of quantum and molecular mechanics, taking into account the ionization states of the amino acid residues in the active site and their interactions with the substrate and competitive inhibitors. The oligonucleotide- and phosphotyrosine-binding cavities important for the inhibitor design have been identified in the enzyme's active site. The developed molecular model allowed us to uncover new Tdp1 inhibitors whose sulfo group is capable of occupying the position of the 3'-phosphate group of the substrate and forming hydrogen bonds with Lys265, Lys495, and other amino acid residues in the phosphotyrosine binding site.

Knockdown of KRT17 by siRNA induces antitumoral effects on gastric cancer cells.

Keratin 17 (KRT17) was shown to be an important molecular marker for predicting the carcinogenesis, progression, and prognosis of various cancer types. Our previous studies identified KRT17 as a possible biomarker for gastric cancer by gene microarray, with an elevated expression that occurred early during tumorigenesis and increased during tumor progression. Based on these findings, we aimed to investigate KRT17 biological functions in gastric adenocarcinoma and its possible use as a rational molecular target for anticancer therapy. We used RNA interference-mediated knockdown of KRT17 expression and analyzed the effects on cell proliferation, cell migration, and signal transduction in two gastric cell lines (AGS and NCI-N87) in vitro and on xenograft growth in vivo. The functional analysis of KRT17 knockdown cell lines showed a decreased cell proliferation (with 42.36%±3.2%) and migration ability (with 37.2%±6.2%) relative to scrambled siRNA control. The in vivo tumorigenicity on nude mice exhibited a significant decrease in tumor weight with 69.14% in xenografts obtained from AGS cells and 84.43% in xeno-NCI-N87 tumors. The analysis on KRT17 knockdown outcome on intracellular signaling identifies AKT/mTOR as the main affected pathway that sustains proliferation and survival, and also the AMPKα1/CREB pathway that was recently shown to induce organ protection and antiinflammatory response. Our results highlight KRT17 as a possible biomarker in gastric cancer promoting tumor growth, motility, and invasion, and suggest that KRT17 can be a valuable molecular target for development of anti-gastric cancer-specific therapies.

The product of the human AHI-1 (Abelson helper integration site) gene: experimental in vitro data point to its involvement in tumor cell invasion.

Each of the steps involved in invasion of tumors requires specific molecular program in which the modulation of adhesive and migratory properties of disseminating cells plays an essential role. The improvement in the knowledge of these mechanisms can lead to discovery of new target candidates in drug development. In this study we focused attention on the product of the human AHI-1 (Abelson helper integration site) gene Jouberin (Jbn). In particular, we explore by in vitro invasion assay, AHI-1 knockdown and electron microscopy, if Jbn is involved in the signaling machinery that regulates tumor invasion. To this purpose tumor cells of different histological derivation (brain, breast, skin) were employed. We found that Jbn expression correlates with the proliferation, invasive potential and invasion strategy of the tested tumor cells, and that its downregulation reduces their capability of migrating and invading the extracellular matrix. The results obtained in this study for the first time point to Jbn as a new candidate involved in the invasion process of tumor cells, and as potential molecular target in anticancer therapy.

PF4 Promotes Platelet Production and Lung Cancer Growth

Co-option of host components by solid tumors facilitates cancer progression and can occur in both local tumor microenvironments and remote locations. Atpresent, the signals involved in long-distance communication remain insufficiently understood. Here, we identify platelet factor 4 (PF4, CXCL4) as an endocrine factor whose overexpression in tumors correlates with decreased overall patient survival. Furthermore, engineered PF4 over-production in a Kras-driven lung adenocarcinoma genetic mouse model expanded megakaryopoiesis in bone marrow, augmented platelet accumulation in lungs, and accelerated de novo adenocarcinogenesis. Additionally, anti-platelet treatment controlled mouse lung cancer progression, further suggesting that platelets can modulate the tumor microenvironment to accelerate tumor outgrowth. These findings support PF4 as a cancer-enhancing endocrine signal that controls discrete aspects of bone marrow hematopoiesis and tumor microenvironment and that should be considered as a molecular target in anticancer therapy.

Expression of chromosomal regional maintenance protein-1 may be associated with subcellular survivin expression in human gastric and colorectal carcinoma.

Survivin, a member of the inhibitor of apoptosis protein family, is a potential prognostic marker and molecular target for anticancer therapies. Chromosomal regional maintenance protein-1 (CRM-1) mediates the nuclear export of proteins such as survivin. The aims of the present study were to compare the expression and subcellular localization of CRM-1 in human gastric and colorectal carcinomas and to assess the association between CRM-1 and survivin expression in these tumor types. The nuclear and cytoplasmic CRM-1 expression rates in gastric carcinoma were 61% (42/69) and 29% (20/69), respectively, while the nuclear and cytoplasmic CRM-1 expression rates in colorectal carcinoma were 55% (43/78) and 37% (29/78), respectively. Nuclear and cytoplasmic CRM-1 expression was found to be significantly correlated with nuclear and cytoplasmic survivin expression in colorectal carcinoma, but not gastric carcinoma. These results indicate that CRM-1 expression patterns differ between gastric and colorectal carcinomas and thus, we hypothesize that CRM-1-mediated nuclear export of survivin may be deregulated in gastric carcinoma. Therefore, CRM-1 may exhibit different functions in gastric and colorectal carcinoma.

Knockdown of USP39 induces cell cycle arrest and apoptosis in melanoma.

The spliceosome machinery composed of multimeric protein complexes guides precursor messenger RNAs (mRNAs) (pre-mRNAs) splicing in eukaryotic cells. Spliceosome components have been shown to be downregulated in cancer and could be a promising molecular target for anticancer therapy. The ubiquitin-specific protease 39 (USP39) is essential for pre-mRNA splicing, and upregulated USP39 expression is noted in a variety of cancers. However, the role of USP39 in the development and progression of melanoma remains unclear. In the present study, USP39 expression was found to be increased in melanoma tissues compared with that in nevus tissues. USP39 silencing via lentivirus-mediated short hairpin RNA (shRNA) significantly suppressed melanoma cell proliferation, induced G0/G1 cell cycle phase arrest, and increased apoptosis in vitro. Moreover, USP39 knockdown suppressed melanoma tumor growth in a xenograft model. In addition, USP39 silencing was associated with the increased expressions of p21, p27, and Bax. Furthermore, the inhibition of USP39 expression decreased the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, indicating that ERK signaling pathways might be involved in the regulation of melanoma cell proliferation by USP39. Our findings suggest that USP39 may play crucial roles in the development and pathogenesis of melanoma, and it may serve as a potential therapeutic target for melanoma.

Actin bundling by dynamin2 and cortactin is implicated in cell migration by stabilizing filopodia in human non-small cell lung carcinoma cells.

The endocytic protein dynamin participates in the formation of actin-based membrane protrusions such as podosomes, pseudopodia, and invadopodia, which facilitate cancer cell migration, invasion, and metastasis. However, the role of dynamin in the formation of actin-based membrane protrusions at the leading edge of cancer cells is unclear. In this study, we demonstrate that the ubiquitously expressed dynamin 2 isoform facilitates cell migration by stabilizing F-actin bundles in filopodia of the lung cancer cell line H1299. Pharmacological inhibition of dynamin 2 decreased cell migration and filopodial formation. Furthermore, dynamin 2 and cortactin mostly colocalized along F-actin bundles in filopodia of serum-stimulated H1299 cells by immunofluorescent and immunoelectron microscopy. Knockdown of dynamin 2 or cortactin inhibited the formation of filopodia in serum-stimulated H1299 cells, concomitant with a loss of F-actin bundles. Expression of wild-type cortactin rescued the punctate-like localization of dynamin 2 and filopodial formation. The incubation of dynamin 2 and cortactin with F-actin induced the formation of long and thick actin bundles, with these proteins colocalizing at F-actin bundles. A depolymerization assay revealed that dynamin 2 and cortactin increased the stability of F-actin bundles. These results indicate that dynamin 2 and cortactin participate in cell migration by stabilizing F-actin bundles in filopodia. Taken together, these findings suggest that dynamin might be a possible molecular target for anticancer therapy.

MicroRNA-19a/b mediates grape seed procyanidin extract-induced anti-neoplastic effects against lung cancer

Oncomirs are microRNAs (miRNA) associated with carcinogenesis and malignant transformation. They have emerged as potential molecular targets for anti-cancer therapy. We hypothesize that grape seed procyanidin extract (GSE) exerts antineoplastic effects through modulations of oncomirs and their downstream targets. We found that GSE significantly down-regulated oncomirs miR-19a and -19b in a variety of lung neoplastic cells. GSE also increased mRNA and protein levels of insulin-like growth factor II receptor (IGF-2R) and phosphatase and tensin homolog (PTEN), both predicted targets of miR-19a and -19b. Furthermore, GSE significantly increased PTEN activity and decreased AKT phosphorylation in A549 cells. Transfection of miR-19a and -19b mimics reversed the up-regulations of IGF2R and PTEN gene expression and abrogated the GSE induced anti-proliferative response. Additionally, oral administration of leucoselect phytosome, comprised of standardized grape seed oligomeric procyanidins complexed with soy phospholipids, to athymic nude mice via gavage, significantly down-regulated miR-19a, -19b and the miR-17-92 cluster host gene (MIR17HG) expressions, increased IGF-2R, PTEN, decreased phosphorylated-AKT in A549 xenograft tumors, and markedly inhibited tumor growth. To confirm the absorption of orally administered GSE, plasma procyanidin B1 levels, between 60 and 90 min after gavage of leucoselect phytosome (400 mg/kg), were measured by LC/MS at week 2 and 8 of treatment; the estimated concentration that was associated with 50% growth inhibition (IC50) (1.3 μg/mL) in vitro was much higher than the IC50 (0.032–0.13 μg/ml) observed in vivo. Our findings reveal novel antineoplastic mechanisms by GSE and support the clinical translation of leucoselect phytosome as an anti-neoplastic and chemopreventive agent for lung cancer.

Inhibition of KIF14 Suppresses Tumor Cell Growth and Promotes Apoptosis in Human Glioblastoma

Background/Aims: The mitotic kinesin superfamily protein KIF14 is essential for cytokinesis and chromosome segregation, and increased KIF14 expression is related to a variety of human cancers. However, the role of KIF14 in the development and malignant progression of astrocytomas and the underlying mechanisms remain unclear. The present study examined the relation between KIF14 and the pathogenesis of malignant astrocytoma. Methods and Results: The role of KIF14 in astrocytoma development and progression was investigated by analyzing KIF14 expression using SYBR Green quantitative real-time RT-PCR, western blotting and immunohistochemistry in human astrocytoma and normal brain tissues. KIF14 expression was higher in astrocytoma samples, and was positively correlated with pathological grade and proliferative activity indicated by Ki-67 staining. SiRNA knockdown of KIF14 inhibited tumor growth in vitro and in vivo, attenuated anchorage-independent growth, and induced G2/M phase arrest, cytokinesis failure and apoptosis in glioblastoma cell lines in association with decreased AKT phosphorylation and activity. Conclusions: The upregulation of KIF14 in astrocytoma is associated with disease severity, and suppression of KIF14 inhibits cell proliferation and induces apoptosis through a mechanism involving the inactivation of AKT signaling, suggesting that KIF14 plays an important role in astrocytoma tumorigenesis and could be a promising molecular target for anticancer therapy.

Abstract LB-11: Discovery, synthesis, and structure-activity relationship of N-benzyl-2-phenylpyrimidin-4-amine derivatives as potent USP1/UAF1 deubiquitinase inhibitors with anticancer activity against non-small cell lung cancer

Abstract Protein ubiquitination is a dynamic and reversible post-translational modification that has been linked to many essential cellular processes in eukaryotes. In addition, deregulation of ubiquitin conjugation or deconjugation has been implicated in the pathogenesis of many human diseases, including cancer. For example, the deubiquitinating enzyme USP1 (ubiquitin-specific protease 1) in association with its WD40-repeat protein binding partner, UAF1 (USP1-associated factor 1), is a known regulator of DNA damage tolerance and repair and has been proposed as a promising target for anticancer therapy. To further evaluate the USP1/UAF1 complex as a therapeutic target, we conducted a quantitative high throughput screen of >400,000 compounds and subsequent medicinal chemistry optimization in pursuit of small molecules that inhibit the deubiquitinating activity of USP1/UAF1. These efforts lead to the identification of ML323 and a series of N-benzyl-2-phenylpyrimidin-4-amine derivatives, which possess nanomolar USP1/UAF1 inhibition and exhibit excellent selectivity over related proteases. Moreover, we demonstrate a strong correlation between compound IC50 values for USP1/UAF1 inhibition in vitro and activity in non-small cell lung cancer cells, specifically increasing the level of ubiquitinated-PCNA and decreasing cell survival. Taken together, our results establish the druggability of the USP1/UAF1 deubiquitinase complex and its potential as molecular target for anticancer therapies. Citation Format: Thomas S. Dexheimer, Andrew S. Rosenthal, Diane Luci, Qin Liang, Mark A. Villamil, Ajit Jadhav, Anton Simeonov, Zhihao Zhuang, David J. Maloney. Discovery, synthesis, and structure-activity relationship of N-benzyl-2-phenylpyrimidin-4-amine derivatives as potent USP1/UAF1 deubiquitinase inhibitors with anticancer activity against non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-11. doi:10.1158/1538-7445.AM2014-LB-11

Abstract 4020: Exploring target gene(s) within chromosome 19-amplification detected in a subclone from metastatic tumors in mouse transplantable OSCC

Abstract Cancer metastasis is caused by a highly complex molecular and cellular mechanism. Although this mechanism has been gradually revealed, there are few genes found as useful diagnostic markers or molecular targets for clinical application so far. In this study, we attempted to establish highly metastatic cells by in vivo selection for identifying metastasis-related genes with an oral squamous cell carcinoma cell line (HOC313-parent). As a result, we established a highly metastatic subline (HOC313-LM) derived from a metastatic site of axillary lymph node. To explore metastasis-related genes and signaling pathways, we performed integrative omics including several genomic and epigenomic approaches combined with systems biology. Since the amplification of chromosome 19 was identified in HOC313-LM by array CGH, we focused on this amplicon containing 44 genes. Among them, one gene, which was essential for the posttranslational modification, was associated with tumor formation and metastasis in vivo via the Rho signaling pathway. Indeed, activation of the Rho signaling pathway emerged in HOC313-LM by our systems biology approach using the gene expression data sets. In addition, the expression pattern of this gene correlated with poor disease-free survival and distant metastasis in patients with esophageal squamous cell carcinoma. Then, we performed therapeutic experiments using a known inhibitor of this gene, and found that tumor growth was inhibited by this compound. Taken together, our findings suggested that this gene might serve as a novel molecular target for anti-cancer therapy. Citation Format: Tomoki Muramatsu, Ken-ichi Kozaki, Seiya Imoto, Rui Yamaguchi, Hitoshi Tsuda, Tatsuyuki Kawano, Satoru Miyano, Johji Inazawa. Exploring target gene(s) within chromosome 19-amplification detected in a subclone from metastatic tumors in mouse transplantable OSCC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4020. doi:10.1158/1538-7445.AM2014-4020

Synthesis and structure-activity relationship studies of N-benzyl-2-phenylpyrimidin-4-amine derivatives as potent USP1/UAF1 deubiquitinase inhibitors with anticancer activity against nonsmall cell lung cancer.

Deregulation of ubiquitin conjugation or deconjugation has been implicated in the pathogenesis of many human diseases including cancer. The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1), in association with UAF1 (USP1-associated factor 1), is a known regulator of DNA damage response and has been shown as a promising anticancer target. To further evaluate USP1/UAF1 as a therapeutic target, we conducted a quantitative high throughput screen of >400000 compounds and subsequent medicinal chemistry optimization of small molecules that inhibit the deubiquitinating activity of USP1/UAF1. Ultimately, these efforts led to the identification of ML323 (70) and related N-benzyl-2-phenylpyrimidin-4-amine derivatives, which possess nanomolar USP1/UAF1 inhibitory potency. Moreover, we demonstrate a strong correlation between compound IC50 values for USP1/UAF1 inhibition and activity in nonsmall cell lung cancer cells, specifically increased monoubiquitinated PCNA (Ub-PCNA) levels and decreased cell survival. Our results establish the druggability of the USP1/UAF1 deubiquitinase complex and its potential as a molecular target for anticancer therapies.

DUSP28 contributes to human hepatocellular carcinoma via regulation of the p38 MAPK signaling.

DUSP28, a member of the atypical dual‑specificity phosphatase (DUSP) family, is a candidate tumor-related gene in hepatocellular carcinoma (HCC) selected by genome‑wide approach, but its pathological role in HCC has not been elucidated. Here, we report for the first time that DUSP28 is involved in HCC progression. Quantitative real‑time PCR and semi‑quantitative RT-PCR showed notably elevated expression of DUSP28 in HCC specimens compared to that in corresponding adjacent non‑tumor liver. DUSP28 overexpression promoted HCC cell proliferation, colony formation and soft agar colony formation in vitro while DUSP28 knockdown resulted in the opposite effects. Furthermore, the flow cytometric analysis indicated that DUSP28 could lead to an increased population of cancer cells in S phase, with a concomitant decrease of cells in G1 phase. Investigation of the mechanism revealed that DUSP28 could activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Taken together, these data demonstrate that DUSP28 plays a significant role in HCC progression and may be a feasible molecular target for anti-cancer therapy.

Role of glycosyltransferase PomGnT1 in glioblastoma progression.

Glioblastoma multiforme (GBM) is the most aggressive and invasive brain tumor, for which novel prognostic markers and predictors of therapeutic response are urgently needed. We reported previously that levels of peptide-O-linked mannose β-1,2-N-acetylglucosaminyltransferase 1 (PomGnT1) in glioma specimens correlated with tumor grade. However, the prognostic significance of PomGnT1 in glioma patients and its function in GBM progression remain unknown. Clinical relevance of PomGnT1 in GBM patients' prognosis was analyzed both in a clinically annotated expression dataset of 446 GBM tumor specimens and in 82 GBM tumor samples collected at our institution. The function of PomGnT1 in glioma growth and invasion, and the underlying mechanisms of PomGnT1 regulation were explored in vitro and in vivo. PomGnT1 expression in GBM tissues was closely associated with poor prognosis in GBM patients. Forced overexpression of PomGnT1 in glioblastoma cells impaired cell adhesion and increased their proliferation and invasion in vitro. Subsequent in vivo experiments showed that overexpression of PomGnT1 promoted tumor growth and shortened the survival time of tumor-bearing mice in an orthotopic model. Conversely, stable short hairpin RNA-mediated knockdown of PomGnT1 expression produced opposite effects both in vitro and in vivo. Mechanistic studies revealed that activation of epidermal growth factor receptor (EGFR) resulted in EGFR/extracellular signal-regulated kinase-dependent upregulation of PomGnT1, downregulation of receptor-type protein tyrosine phosphatase β, and activation of β-catenin pathway signaling. These findings suggest that PomGnT1 promotes GBM progression via activation of β-catenin and may serve as a prognostic factor for glioma patient survival as well as a novel molecular target for anticancer therapy in malignant glioma.

T-type calcium channels blockers as new tools in cancer therapies

T-type calcium channels are involved in a multitude of cellular processes, both physiological and pathological, including cancer. T-type channels are also often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation, migration, and survival. Here, we review the recent literature and discuss the controversies, supporting the role of T-type Ca(2+) channels in cancer cells and the proposed use of channels blockers as anticancer agents. A growing number of reports show that pharmacological inhibition or RNAi-mediated downregulation of T-type channels leads to inhibition of cancer cell proliferation and increased cancer cell death. In addition to a single agent activity, experimental results demonstrate that T-type channel blockers enhance the anticancer effects of conventional radio- and chemotherapy. At present, the detailed biological mechanism(s) underlying the anticancer activity of these channel blockers is not fully understood. Recent findings and ideas summarized here identify T-type Ca(2+) channels as a molecular target for anticancer therapy and offer new directions for the design of novel therapeutic strategies employing channels blockers. Physiological relevance: T-type calcium channels are often aberrantly expressed or deregulated in cancer cells, supporting their proliferation, survival, and resistance to treatment; therefore, T-type Ca(2+) channels could be attractive molecular targets for anticancer therapy.

Metformin inhibits heme oxygenase-1 expression in cancer cells through inactivation of Raf-ERK-Nrf2 signaling and AMPK-independent pathways

Resistance to therapy is the major obstacle to more effective cancer treatment. Heme oxygenase-1 (HO-1) is often highly up-regulated in tumor tissues, and its expression is further increased in response to therapies. It has been suggested that inhibition of HO-1 expression is a potential therapeutic approach to sensitize tumors to chemotherapy and radiotherapy. In this study, we tested the hypothesis that the anti-tumor effects of metformin are mediated by suppression of HO-1 expression in cancer cells. Our results indicate that metformin strongly suppresses HO-1 mRNA and protein expression in human hepatic carcinoma HepG2, cervical cancer HeLa, and non-small-cell lung cancer A549 cells. Metformin also markedly reduced Nrf2 mRNA and protein levels in whole cell lysates and suppressed tert-butylhydroquinone (tBHQ)-induced Nrf2 protein stability and antioxidant response element (ARE)-luciferase activity in HepG2 cells. We also found that metformin regulation of Nrf2 expression is mediated by a Keap1-independent mechanism and that metformin significantly attenuated Raf-ERK signaling to suppress Nrf2 expression in cancer cells. Inhibition of Raf-ERK signaling by PD98059 decreased Nrf2 mRNA expression in HepG2 cells, confirming that the inhibition of Nrf2 expression is mediated by an attenuation of Raf-ERK signaling in cancer cells. The inactivation of AMPK by siRNA, DN-AMPK or the pharmacological AMPK inhibitor compound C, revealed that metformin reduced HO-1 expression in an AMPK-independent manner. These results highlight the Raf-ERK-Nrf2 axis as a new molecular target in anticancer therapy in response to metformin treatment.

Abstract LB-305: Novel Rac1-GEF inhibitors as anticancer agents for aggressive tumors.

Abstract Small GTPases of the Rho family, including Rac1, regulate fundamental cellular processes like actin cytoskeleton reorganization, cell migration and cell cycle progression. Rac1 has been associated with transformation, tumor progression and tumor cell migration in different type of cancers, including aggressive breast cancer and glioma. These proteins cycle between an active, GTP-bound state and an inactive, GDP-bound state. This cycle is highly regulated by many regulatory proteins, including activators such as guanine nucleotide exchange factors (GEFs). We searched for Rac1 inhibitors able to interfere Rac1-GEF interaction using a docking-based virtual library screening approach. We selected ZINC69391 compound. This small-molecule was able to interfere Rac-Tiam1 and Rac-DOCK180 interaction in vitro and reduced Rac1-GTP intracellular levels but had no effect on the closely related Cdc42 GTPase. ZINC69391 also affected the activation of Pak1, one important Rac1 effector. This compound reduced cell migration, inhibited cell proliferation and arrested cell cycle progression in breast cancer cells and glioma cells. We also evaluated this compound in vivo and ZINC69391 was able to reduce 60% lung colonization in a breast cancer experimental metastasis model. We further evaluated novel analogs looking for more potent and specific Rac1 inhibitors. We synthesized a group of novel disubstituted guanidines and we screened these analogs in vitro. One of them, 1A-116 analog, showed to be more potent than the parental compound, reducing cancer cell proliferation and Rac1 activation at the low micromolar range. These data further confirm Rac1 GTPase as a valuable molecular target for anti-cancer therapies and drug development in breast cancer and glioma, where Rac1 presents a relevant role. Citation Format: Georgina A. Cardama, Nazareno Gonzalez, Maria Julieta Comin, Adrian G. Turjanski, Daniel Fernando Alonso, Daniel Eduardo Gomez, Pablo Lorenzano Menna. Novel Rac1-GEF inhibitors as anticancer agents for aggressive tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-305. doi:10.1158/1538-7445.AM2013-LB-305

Computational study of the effects of protein tyrosine nitrations on the catalytic activity of human thymidylate synthase

Tyrosine nitration is a widespread post-translational modification capable of affecting both the function and structure of the host protein molecule. Enzyme thymidylate synthase (TS), a homodimer, is a molecular target for anticancer therapy. Recently purified TS preparations, isolated from mammalian tissues, were found to be nitrated, suggesting this modification to appear endogenously in normal and tumor tissues. Moreover, human TS (hTS) nitration in vitro led to a by twofold lowered catalytic activity following nitration in average of 1 tyrosine residue per monomer (Dąbrowska-Maś et al. in Org Biomol Chem 10:323-331, 2012), with the modification identified by mass spectrometry at seven different sites (Y33, Y65, Y135, Y213, Y230, Y258 and Y301). In the present paper, combined computational approach, including molecular and essential dynamics and free energy computations, was used to predict the influence on the activity of hTS of nitration of each of the seven tyrosine residues. The simulations were based on the crystal structure of hTS ternary complex with dUMP and Tomudex (PDB code: 1I00), with the Tomudex molecule replaced by the molecule of TS cofactor analogue, tetrahydrofolate. The present results indicate that while with nitration of five out of seven residues (Y33, Y135, Y230, Y258 and Y301), single residue modification appears to have a strong reducing effect on the activity, with the remaining two, Y65 and Y213, no or a weaker influence is apparent. Taken together, these results demonstrate that tyrosine nitrations in the hTS enzyme show clear tendency to influence the structure and dynamics and, in turn, catalytic properties of the host enzyme. These effects are overall distance-dependent.

Efficacy of everolimus, a novel mTOR inhibitor, against basal‐like triple‐negative breast cancer cells

Patients with triple-negative breast cancers (TNBCs) typically have a poor prognosis because such cancers have no effective therapeutic targets, such as estrogen receptors for endocrine therapy or human epidermal growth factor receptor 2 (HER2) receptors for anti-HER2 therapy. As the phosphatidylinositol 3' kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) cascade is activated in TNBCs, mTOR is a potential molecular target for anticancer therapy. In this study, we investigated the antitumor activities of everolimus, an oral mTOR inhibitor, in nine TNBC cell lines. Everolimus effectively inhibited cell growth at concentrations under 100 nM (IC(50)) in five cell lines and even in the 1-nM range in three of the five cell lines. To identify specific characteristics that could be used as predictive markers of efficacy, we evaluated the expressions of proteins in the mTOR cascade, basal markers, and cancer stem cell markers using western blotting, fluorescent in situ hybridization (FISH), or immunohistochemistry. All five of the sensitive cell lines were categorized as a basal-like subtype positive for either epidermal growth factor receptor (EGFR) or CK5/6, although resistant cell lines were not of this subtype and tended to exhibit the characteristics of cancer stem cells, with decreased E-cadherin and the increased expression of Snail or Twist. In vivo assays demonstrated antitumor activity in a mouse xenograft model of basal-like breast cancer, rather than non-basal breast cancer. These results suggest that everolimus has favorable activity against basal-like subtypes of TNBCs. Epidermal growth factor receptor and CK5/6 are positive predictive markers of the TNBC response to everolimus, while cancer stem cell markers are negative predictive markers.

An Ultrahigh Affinity d-Peptide Antagonist Of MDM2

The oncoprotein MDM2 negatively regulates the activity and stability of the p53 tumor suppressor and is an important molecular target for anticancer therapy. Aided by mirror image phage display and native chemical ligation, we have previously discovered several proteolysis-resistant duodecimal d-peptide antagonists of MDM2, termed (D)PMI-α, β, γ. The prototypic d-peptide inhibitor (D)PMI-α binds ((25-109))MDM2 at an affinity of 220 nM and kills tumor cells in vitro and inhibits tumor growth in vivo by reactivating the p53 pathway. Herein, we report the design of a superactive d-peptide antagonist of MDM2, termed (D)PMI-δ, of which the binding affinity for ((25-109))MDM2 has been improved over (D)PMI-α by 3 orders of magnitude (K(d) = 220 pM). X-ray crystallographic studies validate (D)PMI-δ as an exceedingly potent inhibitor of the p53-MDM2 interaction, promising to be a highly attractive lead drug candidate for anticancer therapeutic development.