New Strategy For Anticancer Therapy Research Articles

Abstract 2893: Inhibition of ADAMTS4 and 5 to regulate the tumor microenvironment

Abstract Background: Triple-negative breast cancer accounts for approximately 10~15% of all breast cancers and is characterized by the absence of ER, PR, and HER2 receptors, making targeted therapies challenging due to the lack of biomarkers. ADAMTSs (ADAM metallopeptidase with thrombospondin) are complex extracellular proteases known for their roles in facilitating cancer cell proliferation and safeguarding tumor functions. This enzyme is secreted by cancer cells and various immune cells, influencing the tumor microenvironment. ADAMTSs modulate cellular adhesion, migration, proliferation, and vascular development by cleaving components of the extracellular matrix and regulatory factors such as chemokines and cytokines. Our aim is to positively manipulate the tumor microenvironment of triple-negative breast cancer by inhibiting ADAMTSs, presenting a new strategy for treating this challenging type of breast cancer that lacks targeted therapeutic options. Results: To confirm the anticancer effects of a protease inhibitor concurrently targeting ADAMTS4 and 5, a co-culture of triple-negative breast cancer cells (MDA-MB-157) and THP-1 was established to artificially create a tumor microenvironment. Subsequently, a wound healing assay was conducted, revealing that treatment with the ADAMTS4 and 5 dual inhibitor effectively inhibited cancer cell migration. This observed anticancer effect is attributed to the inhibition of CXCL16 secretion by the ADAMTS4 and 5 dual inhibitor. To verify the anti-tumor mechanisms of ADAMTS4 and 5, MDA-MB-157 cells, a triple-negative breast cancer cell line, were treated with IFNγ and TNFα to promote CXCL16 secretion artificially. Upon treatment with varying concentrations of the ADAMTS4,5 dual inhibitor, it was confirmed that the secretion of CXCL16 was inhibited in a dose-dependent manner. In a mouse model with transplanted 4T1 breast cancer cells, treatment with the ADAMTS4,5 dual inhibitor resulted in the suppression of CXCL16 secretion, leading to an increase in M1 macrophages. Additionally, it was observed that the inhibitor induced CD8+ T cell activation. These regulation and changes of immune cells through inhibition of ADAMTS4 and 5 control the TME to resist tumor cells. Conclusion: ADAMTSs are known as protein-degrading enzymes responsible for the degradation, modification, and regulation of proteins. Recent research has revealed that ADAMTSs play a crucial role in the proliferation and survival of cancer. Activation of ADAMTS4 and 5 has been shown to promote the secretion of CXCL16, leading to the differentiation of macrophages into tumor-associated macrophages. Specifically, ADAMTS4 and 5 convert membrane-bound CXCL16 into a soluble form, exacerbating the tumor microenvironment of triple-negative breast cancer. Therefore, inhibiting ADAM4,5 is intended to change or maintain TME in a suitable state for anti-cancer therapy, which could lead to a new strategy for anti-cancer therapy. Citation Format: Cham han Lee, Seong Keun Kim, Sung Moo Kim, Young Sang Kim, Sang Hyeob Lee, Seong Yun Ha, Yun Sun Lee, Jin Yi Yang. Inhibition of ADAMTS4 and 5 to regulate the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2893.

Discovering selective antiferroptotic inhibitors of the 15LOX/PEBP1 complex noninterfering with biosynthesis of lipid mediators

Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis invitro and invivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators invivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.

Natural products targeting glycolytic signaling pathways-an updated review on anti-cancer therapy.

Glycolysis is a complex metabolic process that occurs to convert glucose into pyruvate to produce energy for living cells. Normal cells oxidized pyruvate into adenosine triphosphate and carbon dioxide in the presence of oxygen in mitochondria while cancer cells preferentially metabolize pyruvate to lactate even in the presence of oxygen in order to maintain a slightly acidic micro-environment of PH 6.5 and 6.9, which is beneficial for cancer cell growth and metastasis. Therefore targeting glycolytic signaling pathways provided new strategy for anti-cancer therapy. Natural products are important sources for the treatment of diseases with a variety of pharmacologic activities. Accumulated studies suggested that natural products exhibited remarkable anti-cancer properties both in vitro and in vivo. Plenty of studies suggested natural products like flavonoids, terpenoids and quinones played anti-cancer properties via inhibiting glucose metabolism targets in glycolytic pathways. This study provided an updated overview of natural products controlling glycolytic pathways, which also provide insight into druggable mediators discovery targeting cancer glucose metabolism.

Abstract 1583: SEA-TGT is an empowered anti-TIGIT antibody that displays superior combinatorial activity with several therapeutic agents

Abstract TIGIT is an immunoregulatory receptor expressed on activated and memory T cells, Tregs, and NK cells. TIGIT binding to CD155 and CD112 on tumor cells drives an inhibitory signal resulting in decreased T cell functionality. TIGIT targeting has been reported to release these inhibitory signals, drive Treg depletion, augment CD8+ T cell generation, and promote anti-tumor responses. Anti-TIGIT therapies have come to prominence recently as new strategies for anti-cancer therapy particularly in combination with other immune checkpoint inhibitors because of their complementary mechanisms of action. We have recently developed an investigational anti-TIGIT monoclonal antibody that employs our proprietary sugar engineered antibody (SEA) technology to create a nonfucosylated antibody, termed SEA-TGT. In the present studies, we have expanded our investigation of SEA-TGT and compared it to other antibody formats to understand how differences in complementarity-determining regions (CDR) and backbone effect both in vitro activity and in vivo anti-tumor response. We also investigated how these differences affect the ability of anti-TIGIT mAbs to combine with other anti-tumor therapies. The nonfucosylated SEA-TGT demonstrates a unique activating to inhibitory FcγR binding profile. SEA-TGT demonstrates enhanced FcγRIIIA binding with concomitant reduction in inhibitory FcγRIIb receptor binding. This binding with SEA-TGT was distinct from standard IgG1 backbones and gains effector function modifications compared to other anti-TIGIT mAbs. The differential ability to engage activating vs. inhibitory Fcγ receptors translated into distinct differences in preclinical activity. The differences seen in vitro were recapitulated in vivo as SEA-TGT led to enhanced tumor activity in syngeneic tumor models. Anti-TIGIT treatments are being pursued clinically in combination with other therapeutic modalities, as such we evaluated treatment with the TIGIT mAbs with different effector function backbones with various other anti-tumor therapies. When used at sub-therapeutic doses, SEA-TGT exhibited greatly enhanced anti-tumor activity in combination with both checkpoint therapy as well as with an ICD inducing vc-MMAE antibody-drug conjugate. Collectively, these data continue to strengthen our findings that the nonfucosylated effector function enhanced backbone of SEA-TGT confers superior preclinical anti-tumor activity as a monotherapy agent and in combination with PD(L)1 agents and vc-MMAE ADCs. We have initiated a phase 1 trial testing the safety and activity of SEA-TGT alone and in combination with anti-PD-1 therapy in patients with advanced solid tumors and select lymphomas. Citation Format: Alyson Smith, Weiping Zeng, Sasha Lucas, Bryan Grogan, Amber Blackmarr, Serena Wo, Scott Peterson, Shyra Gardai. SEA-TGT is an empowered anti-TIGIT antibody that displays superior combinatorial activity with several therapeutic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1583.

Wnt5a-induced docking of Plk1 on HEF1 promotes HEF1 translocation and tumorigenesis.

Upregulation of human enhancer filamentation 1 (HEF1/NEDD9/Cas-L) and Polo-like kinase 1 (Plk1) is closely correlated with metastasis of human cancer. However, the mechanism by which the overexpression of HEF1 or Plk1 stimulates cancer metastasis and induces tumorigenesis remains enigmatic. In addition, the accumulation of HEF1 at the focal adhesion (FA) is known to be an essential event in cancer cell migration, but the mechanism of how HEF1 is targeted to the FA remains yet to be unveiled. This study was performed to elucidate the FA docking mechanism of HEF1 and to determine its effect on tumorigenesis. To confirm the effect of the kinase on HEF1 translocation, various expression-knockdown stable cell lines were generated using a lentivirus system, and the effect of the HEF1-Plk1 complex on tumorigenesis was confirmed using a xenograft mouse model. Here, we show that Wnt5a-dependent Plk1 binding to HEF1 is critically required for HEF1 translocation to the FA. We also confirmed that Plk1 and CK1δ activities essential for HEF1 translocation are induced by Wnt5a. Finally, we confirmed the induction of tumorigenesis by the HEF1-Plk1 complex in the xenograft mouse model. Our data collectively unveil the Wnt5a-CK1δ-HEF1-Plk1-FA remodeling pathway that governs HEF1 transportation to the FA to induce cell migration and tumorigenesis. This study sheds light on a mechanism underlying tumorigenesis and provides new strategies for anticancer therapy.

High blood flow shear stress values are associated with circulating tumor cells cluster disaggregation in a multi-channel microfluidic device.

Metastasis represents a dynamic succession of events involving tumor cells which disseminate through the organism via the bloodstream. Circulating tumor cells (CTCs) can flow the bloodstream as single cells or as multicellular aggregates (clusters), which present a different potential to metastasize. The effects of the bloodstream-related physical constraints, such as hemodynamic wall shear stress (WSS), on CTC clusters are still unclear. Therefore, we developed, upon theoretical and CFD modeling, a new multichannel microfluidic device able to simultaneously reproduce different WSS characterizing the human circulatory system, where to analyze the correlation between SS and CTC clusters behavior. Three physiological WSS levels (i.e. 2, 5, 20 dyn/cm2) were generated, reproducing values typical of capillaries, veins and arteries. As first validation, triple-negative breast cancer cells (MDA-MB-231) were injected as single CTCs showing that higher values of WSS are correlated with a decreased viability. Next, the SS-mediated disaggregation of CTC clusters was computationally investigated in a vessels-mimicking domain. Finally, CTC clusters were injected within the three different circuits and subjected to the three different WSS, revealing that increasing WSS levels are associated with a raising clusters disaggregation after 6 hours of circulation. These results suggest that our device may represent a valid in vitro tool to carry out systematic studies on the biological significance of blood flow mechanical forces and eventually to promote new strategies for anticancer therapy.

HHQ-4, a quinoline derivate, preferentially inhibits proliferation of glucose-deprived breast cancer cells as a GRP78 down-regulator

As a central regulator for endoplasmic reticulum (ER) stress, glucose-regulated protein 78 (GRP78), controls the activation of ER-transmembrane signaling mechanisms by inducing unfolded protein response (UPR) in response to ER stress. Although limited glucose availability often occurs in poorly vascularized solid cancers, cancer cells often initiate the UPR to support cellular homeostasis and survival under stress conditions. Therefore, targeting GRP78 expression and UPR pathway activation may provide a new strategy for anticancer therapy. Based on this premise, we investigated the molecular mechanisms of a synthetic quinolone derivative, 2-hexyl-3-methyl-4(1H)-quinolinone (HHQ-4), in regulating the GRP78 expression and UPR transcriptional program under glucose deprivation or 2-deoxy-d-glucose (2-DG)-stressed conditions. We found that HHQ-4 suppressed the transcriptional and translational expression of GRP78 gene in glucose-deprived breast cancer cells. HHQ-4 also showed selective antiproliferative activity against glucose-deprived breast cancer cells. Constitutive expression of GRP78 completely prevented breast cancer cells from HHQ-4-mediated proliferation inhibition during glucose starvation, stressing the important role of suppression of the GRP78 in HHQ-4-mediated cell proliferation inhibition. HHQ-4 was also found to exert inhibitory activity against breast cancer cell proliferation by suppressing three survival arms of the UPR, including PERK/eIF2α/ATF4, IRE1/XBP1, and ATF6, which orchestrate an intricate signaling network to modulate GRP78 gene transcription under glucose-deprived stress. Furthermore, HHQ-4 combined with 2-DG synergistically inhibited breast cancer cell proliferation. Our findings show HHQ-4 could be a promising candidate, alone or in combination with 2-DG, for selectively inhibiting breast cancer cell proliferation by down-regulating the transcription and expression of GRP78 under stressful microenvironments.

Diverse autophagy and apoptosis in myeloid leukemia cells induced by 20(s)-GRh2 and blue LED irradiation.

Autophagy is an important mechanism for cell death regulation. To improve the anticancer effect during the treatment of leukemia and promote the apoptosis of leukemic cells, it is important to define the relationship between autophagy and apoptosis. A key bioactive compound in traditional Chinese medicine, 20(s)-Ginsenoside (GRh2), demonstrated an advancement in leukemia treatment. Blue LED therapy (BL) is a physical treatment method that can induce leukemic cell death. In this study, we tested the effect of 20(s)-GRh2, BL, and their combination (BL–GRh2) on the activation of leukemic cell apoptosis and autophagy. Both treatments, whether used individually or simultaneously, induce apoptosis through the induction of reactive oxygen species (ROS), disrupted mitochondrial membrane potential (MMP) and regulated the expression of apoptosis-related genes and proteins. Furthermore, using western blotting to analyze the autophagy markers LC3B and P62, we detected the activation of autophagy. In cells treated with autophagy inhibitor 3-MA, both autophagy and apoptosis were inhibited, either by BL alone or by BL–GRh2. However, apoptosis in 20(s)-GRh2-treated cells was enhanced. In cells treated with apoptosis suppressor Z-VAD-FMK, autophagy was inhibited in the BL and BL–GRh2-treated cells, although it was enhanced in cells treated with 20(s)-GRh2 alone. Moreover, we observed a stronger induction of apoptosis by BL–GRh2 in myeloid leukemia cells. Our data indicate that autophagy induced by different factors can play diverse roles on the same cells. Our results also indicate that the combination of traditional Chinese medicine with physical therapy may be a new strategy for anti-cancer therapy.

Prognostic and clinical significance of metastasis-associated gene 1 overexpression in solid cancers: A meta-analysis.

Background:In the past 2 decades, metastasis-associated gene 1 (MTA1) has attracted attention for its close association with cancer progression and its roles in chromatin remodeling processes, making it a central gene in cancer. The present meta-analysis was performed to assess MTA1 expression in solid tumors.Materials and methods:This analysis identified studies that evaluated the relationship between MTA1 expression and clinical characteristics or prognosis of patients with solid tumors via the PubMed, Cochrane Library, and Embase electronic databases. Fixed-effect and random-effect meta-analytical techniques were used to correlate MTA1 expression with outcome measures. The outcome variables are shown as odds ratio (OR) or hazard ratio (HR) with 95% confidence interval (CI).Results:Analysis of 40 cohort studies involving 4564 cancer patients revealed a significant association of MTA1 overexpression with tumor patient age (>50 vs. <50 years: combined OR 0.73, 95% CI 0.57–0.94), tumor grade (G3/4 vs. G1/2: combined OR 1.94, 95% CI 1.48–2.53), tumor size (>3 cm vs. <3 cm: combined OR 2.35, 95% CI 1.73–3.19), T stage (T3/4 vs. T1/2: combined OR 2.11, 95% CI 1.74–2.56), lymph node metastasis (yes vs. no: combined OR 2.92, 95% CI 2.26–3.75), distant metastasis (yes vs. no: combined OR 2.26, 95% CI 1.42–3.59), TNM stage (III/IV vs. I/II: combined OR 2.50, 95% CI 1.84–3.38), vascular invasion (yes vs. no: combined OR 2.26, 95% CI 1.92–3.56), and poor overall survival time (HR 1.83; 95% CI: 1.53–2.20; P = .000).Conclusions:Our analyses demonstrate that MTA1 was an effective predictor of a worse prognosis in tumor patients. Moreover, MTA1 may play important role in tumor progression and outcome, and targeting MTA1 may be a new strategy for anti-cancer therapy.

Silver/silver chloride nanoparticles inhibit the proliferation of human glioblastoma cells

Glioblastomas (GBM) are aggressive brain tumors with very poor prognosis. While silver nanoparticles represent a potential new strategy for anticancer therapy, the silver/silver chloride nanoparticles (Ag/AgCl-NPs) have microbicidal activity, but had not been tested against tumor cells. Here, we analyzed the effect of biogenically produced Ag/AgCl-NPs (from yeast cultures) on the proliferation of GBM02 glioblastoma cells (and of human astrocytes) by automated, image-based high-content analysis (HCA). We compared the effect of 0.1-5.0µgmL-1 Ag/AgCl-NPs with that of 9.7-48.5µgmL-1 temozolomide (TMZ, chemotherapy drug currently used to treat glioblastomas), alone or in combination. At higher concentrations, Ag/AgCl-NPs inhibited GBM02 proliferation more effectively than TMZ (up to 82 and 62% inhibition, respectively), while the opposite occurred at lower concentrations (up to 23 and 53% inhibition, for Ag/AgCl-NPs and TMZ, respectively). The combined treatment (Ag/AgCl-NPs + TMZ) inhibited GBM02 proliferation by 54-83%. Ag/AgCl-NPs had a reduced effect on astrocyte proliferation compared with TMZ, and Ag/AgCl-NPs + TMZ inhibited astrocyte proliferation by 5-42%. The growth rate and population doubling time analyses confirmed that treatment with Ag/AgCl-NPs was more effective against GBM02 cells than TMZ (~ 67-fold), and less aggressive to astrocytes, while Ag/AgCl-NP + TMZ treatment was no more effective against GBM02 cells than Ag/AgCl-NPs monotherapy. Taken together, our data indicate that 2.5µgmL-1 Ag/AgCl-NPs represents the safest dose tested here, which affects GBM02 proliferation, with limited effect on astrocytes. Our findings show that HCA is a useful approach to evaluate the antiproliferative effect of nanoparticles against tumor cells.

Emerging role of DUBs in tumor metastasis and apoptosis: Therapeutic implication.

Malfunction of ubiquitin-proteasome system is tightly linked to tumor formation and tumor metastasis. Targeting the ubiquitin-pathway provides a new strategy for anti-cancer therapy. Despite the parts played by ubiquitin modifiers, removal of ubiquitin from the functional proteins by the deubiquitinating enzymes (DUBs) plays an important role in governing the multiple steps of the metastatic cascade, including local invasion, dissemination, and eventual colonization of the tumor to distant organs. Both deregulated ubiquitination and deubiquitination could lead to dysregulation of various critical events and pathways such as apoptosis and epithelial-mesenchymal transition (EMT). Recent TCGA study has further revealed the connection between mutations of DUBs and various types of tumors. In addition, emerging drug design targeting DUBs provides a new strategy for anti-cancer therapy. In this review, we will summarize the role of deubiquitination and highlight the recent discoveries of DUBs with regards to multiple metastatic events including anti-apoptosis pathway and EMT. We will further discuss the regulation of deubiquitination as a novel strategy for anti-cancer therapy.

Knock-down of Pre-mRNA Splicing Factor Prp19 Causes Chromosome Misalignment and Prometaphase Arrest

To verify the role of the newly identified mitotic regulator candidate pre-mRNA processing factor 19 (Prp19) in mitosis and to clarify its underlying mechanism. FACS analyses with propidium iodide (PI) staining were performed to evaluate the effect of Prp19 knockdown on cell cycle distribution. To further clarify the role of Prp19 in mitosis, the effect of Prp19 depletion was monitored by time-lapse imaging of HeLa/GFP-H2B cells. Cold treatment experiment was used to examine the effect of Prp19 knockdown on the attachment of microtubules and kinetochores. To evaluate the effect of Prp19 knockdown on cell apoptosis, the control and Prp19-knockdown cells were analyzed by FACS with annexin V-FITC/-PI double staining. Furthermore, Western blot analysis of cleaved caspase-3 and PARP was also performed. Prp19 knockdown causesd mitotic arrest. Time-lapse imaging analysis showed that depletion of Prp19 in HeLa cells results in prometaphase arrest and chromosome misalignment. Cold treatment experiment showed that attachment between kinetochore and microtubule was impaired by Prp19 knockdown. Moreover, the depletion of Prp19 leaded to cell apoptosis in cancer cells. Prp19 is a key regulator of mitotic progression, and its inhibition may provide a new strategy for anti-cancer therapy.

Evodiamine Induces Apoptosis and Enhances TRAIL-Induced Apoptosis in Human Bladder Cancer Cells through mTOR/S6K1-Mediated Downregulation of Mcl-1

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anti-cancer agents, has been considered as a new strategy for anti-cancer therapy. In this study, we demonstrated that evodiamine, a quinolone alkaloid isolated from the fruit of Evodia fructus, induced apoptosis and enhanced TRAIL-induced apoptosis in human bladder cancer cells. To elucidate the underlying mechanism, we found that evodiamine significantly reduced the protein levels of Mcl-1 in 253J and T24 bladder cancer cells, and overexpression of this molecule attenuated the apoptosis induced by evodiamine alone, or in combination with TRAIL. Further experiments revealed that evodiamine did not affect the mRNA level, proteasomal degradation and protein stability of Mcl-1. On the other hand, evodiamine inhibited the mTOR/S6K1 pathway, which usually regulates protein translation; moreover, knockdown of S6K1 with small interfering RNA (siRNA) effectively reduced Mcl-1 levels, indicating evodiamine downregulates c-FLIP through inhibition of mTOR/S6K1 pathway. Taken together, our results indicate that evodiamine induces apoptosis and enhances TRAIL-induced apoptosis possibly through mTOR/S6K1-mediated downregulation of Mcl-1; furthermore, these findings provide a rationale for the combined application of evodiamine with TRAIL in the treatment of bladder cancer.

Abstract 5628: Overcoming cetuximab resistance in HNSCC: the role of AURKB and DUSP6.

Abstract Introduction: As the epidermal growth factor receptor (EGFR) is expressed in up to 90% of all HNSCC tumors and initiates important signaling pathways in carcinogenesis, cetuximab, an EGFR targeting agent, is a promising therapeutic agent. However, many tumors remain non-responsive. Therefore, unraveling the underlying mechanism of resistance is of major importance. Previously, we performed gene expression studies of cetuximab sensitive (LICR-HN2 and SC263) and cetuximab resistant (LICR-HN1 and Cal27) cell lines. These results indicated an upregulation of Aurora kinase B (AURKB) in LICR-HN1 cells and a downregulation of dual specificity phosphatase 6 (DUSP6) in Cal27 cells. Methods: Growth inhibitory experiments for barasertib, an AURKB targeting agent, and apigenin, a MAPK inhibitor, were performed as single-agent (barasertib: 0-25nM and apigenin: 0-40μM), and in combination with 15nM cetuximab for 168h using simultaneous and sequential treatment schedules. Induction of apoptotic cell death was investigated in LICR-HN1 cells using the Annexin V-FITC apoptosis detection kit for all treatment schedules. Immunohistochemical staining for AURKB was performed on formalin fixed, paraffin-embedded tissues from 52 newly diagnosed HNSCC patient samples, using an automated IHC staining platform and will be correlated with clinicopathological parameters. The phosphorylation status of ERK was investigated using the NanoPro technology and the methylation status of DUSP6 will be analyzed using the Pyrosequencing method. Results: The mitosis-targeting drug barasertib induced cytotoxicity in the cetuximab resistant HNSCC cell lines, Cal27 and LICR-HN1, with IC50 values of 13.15 ± 0.60nM and 17.6 ± 1.38nM, respectively. In combination therapy with cetuximab, no additional benefit over barasertib as single agent was observed. However, preliminary results showed that pretreatment of LICR-HN1 cells with 15nM cetuximab followed by barasertib (10 or 20nM) resulted in less viable cells compared to cetuximab or barasertib treatment alone. AURKB expression was present in HNSCC patients, with intensities ranging from 0 - 100%. Apigenin induced cytotoxicity in the two cetuximab resistant cell lines, Cal27 and LICR-HN1, with IC50 values of 22.29 ± 0.60μM and 30.66 ± 0.97μM respectively. Preliminary results showed that the combination of cetuximab with apigenin had a synergistic effect in the Cal27 cell line. Furthermore, increased phosphorylation of ERK was present in the cetuximab-resistant cell lines compared to the cetuximab-sensitive cell lines. Conclusion: These results seem to confirm that overexpression of AURKB and/or downregulation of DUSP6 are involved in mechanisms of cetuximab resistance in HNSCC cell lines and inhibition of these pathways might, therefore, be a new strategy for anticancer therapy. Citation Format: Carolien Boeckx, Vanessa Deschoolmeester, An Wouters, Ken Op de Beeckx, Patrick Pauwels, Olivier Vanderveken, Guy Van Camp, Wim Vanden Berghe, Pol Specenier, Jan B. Vermorken, Marc Peeters, Marc Baay, Filip Lardon. Overcoming cetuximab resistance in HNSCC: the role of AURKB and DUSP6. [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 5628. doi:10.1158/1538-7445.AM2013-5628

Design of pH responsive clickable prodrugs applied to histone deacetylase inhibitors: A new strategy for anticancer therapy

The aim of this study was to develop clickable prodrugs bearing a tunable pH responsive linker designed for acidic pH-mediated release of histone deacetylase inhibitors. HDACi are an important class of molecules belonging to the epigenetic modulators used for innovative cancer strategies. The behavior of these prodrugs was determined by a bioluminescence resonance energy transfer assay in living tumor cells. This work demonstrated that this innovative type of clickable prodrugs entered cancer cells and showed restored anti proliferative properties attributed to the effective release of the HDAC inhibitors. A correlation between kinetic studies, dose responses, and biological activities was obtained, making such clickable prodrugs good candidates for new strategies in epigenetic-oriented anticancer therapies.

TRPC Channels and Their Splice Variants are Essential for Promoting Human Ovarian Cancer Cell Proliferation and Tumorigenesis

TRPC channels are Ca²⁺-permeable cationic channels controlling Ca²⁺ influx response to the activation of G protein-coupled receptors and protein tyrosine kinase pathways or the depletion of Ca²⁺ stores. Here we aimed to investigate whether TRPC can act as the potential therapeutic targets for ovarian cancer. The mRNAs of TRPC1, TRPC3, TRPC4 and TRPC6 were detected in human ovarian adenocarcinoma. The spliced variants of TRPC1β, TRPC3a, TRPC4β, TRPC4γ, and TRPC6 with exon 3 and 4 deletion were highly expressed in the ovarian cancer cells, and a novel spliced isoform of TRPC1 with exon 9 deletion (TRPC1(E9del)) was identified. TRPC proteins were also detected by Western blotting and immunostaining. The expression of TRPC1, TRPC3, TRPC4 and TRPC6 was significantly lower in the undifferentiated ovarian cancer cells, but all-trans retinoic acid up-regulated the gene expression of TRPCs. The expression level was correlated to the cancer differentiation grade. The non-selective TRPC channel blockers, 2-APB and SKF-96365, significantly inhibited the cell proliferation, whilst the increase of TRPC channel activity by trypsin promoted the cell proliferation. Transfection with siRNA targeting TRPC1, TRPC3, TRPC4 and TRPC6 or application of specific blocking antibodies targeting to TRPC channels inhibited the cell proliferation. On the contrary, overexpression of TRPC1, TRPC1(E9del), TRPC3, TRPC4, and TRPC6 increased the cancer cell colony growth. These results suggest that TRPCs and their spliced variants are important for human ovarian cancer development and alteration of the expression or activity of these channels could be a new strategy for anticancer therapy.

TRPC Channels and Their Splice Variants are Essential for Promoting Human Ovarian Cancer Cell Proliferation and Tumorigenesis

TRPC channels are Ca2+-permeable cationic channels controlling Ca2+ influx response to the activation of G protein-coupled receptors and protein tyrosine kinase pathways or the depletion of Ca2+ stores. Here we aimed to investigate whether TRPC can act as the potential therapeutic targets for ovarian cancer. The mRNAs of TRPC1, TRPC3, TRPC4 and TRPC6 were detected in human ovarian adenocarcinoma. The spliced variants of TRPC1β, TRPC3a, TRPC4β, TRPC4γ, and TRPC6 with exon 3 and 4 deletion were highly expressed in the ovarian cancer cells, and a novel spliced isoform of TRPC1 with exon 9 deletion (TRPC1E9del) was identified. TRPC proteins were also detected by Western blotting and immunostaining. The expression of TRPC1, TRPC3, TRPC4 and TRPC6 was significantly lower in the undifferentiated ovarian cancer cells, but all-trans retinoic acid up-regulated the gene expression of TRPCs. The expression level was correlated to the cancer differentiation grade. The non-selective TRPC channel blockers, 2-APB and SKF-96365, significantly inhibited the cell proliferation, whilst the increase of TRPC channel activity by trypsin promoted the cell proliferation. Transfection with siRNA targeting TRPC1, TRPC3, TRPC4 and TRPC6 or application of specific blocking antibodies targeting to TRPC channels inhibited the cell proliferation. On the contrary, overexpression of TRPC1, TRPC1E9del, TRPC3, TRPC4, and TRPC6 increased the cancer cell colony growth. These results suggest that TRPCs and their spliced variants are important for human ovarian cancer development and alteration of the expression or activity of these channels could be a new strategy for anticancer therapy. Keywords: Calcium channels, ovarian cancer, proliferation, SKOV3 cells, TRPC

TRPC Channels and Their Splice Variants are Essential for Promoting Human Ovarian Cancer Cell Proliferation and Tumorigenesis

TRPC channels are Ca²⁺-permeable cationic channels controlling Ca²⁺ influx response to the activation of G protein-coupled receptors and protein tyrosine kinase pathways or the depletion of Ca²⁺ stores. Here we aimed to investigate whether TRPC can act as the potential therapeutic targets for ovarian cancer. The mRNAs of TRPC1, TRPC3, TRPC4 and TRPC6 were detected in human ovarian adenocarcinoma. The spliced variants of TRPC1β, TRPC3a, TRPC4β, TRPC4γ, and TRPC6 with exon 3 and 4 deletion were highly expressed in the ovarian cancer cells, and a novel spliced isoform of TRPC1 with exon 9 deletion (TRPC1(E9del)) was identified. TRPC proteins were also detected by Western blotting and immunostaining. The expression of TRPC1, TRPC3, TRPC4 and TRPC6 was significantly lower in the undifferentiated ovarian cancer cells, but all-trans retinoic acid up-regulated the gene expression of TRPCs. The expression level was correlated to the cancer differentiation grade. The non-selective TRPC channel blockers, 2-APB and SKF-96365, significantly inhibited the cell proliferation, whilst the increase of TRPC channel activity by trypsin promoted the cell proliferation. Transfection with siRNA targeting TRPC1, TRPC3, TRPC4 and TRPC6 or application of specific blocking antibodies targeting to TRPC channels inhibited the cell proliferation. On the contrary, overexpression of TRPC1, TRPC1(E9del), TRPC3, TRPC4, and TRPC6 increased the cancer cell colony growth. These results suggest that TRPCs and their spliced variants are important for human ovarian cancer development and alteration of the expression or activity of these channels could be a new strategy for anticancer therapy.

Binding and intracellular routing of the plant-toxic lectins, lanceolin and stenodactylin

Background The present research studied the interaction of two ribosome-inactivating proteins (RIPs) from Adenia genus with HeLa cells. Namely, lanceolin and stenodactylin were examined in comparison to volkensin, another toxic two-chain RIP from Adenia genus. Methods The binding, endocytosis, intracellular routing, degradation and exocytosis were investigated by measuring the distribution of radiolabelled RIP and by determining its cytotoxicity. Results Stenodactylin was the most toxic, resulting in the greater inhibition of protein synthesis and cell death. Lanceolin and stenodactylin bound to cells with comparable affinity and have a similar number of binding sites (10 5/cell). The uptake of lanceolin and stenodactylin was 13 and 36 times greater, respectively, than that reported for volkensin. The two toxins bound to cell membrane receptors via their lectin B chain, were endocytosed through a clathrin-independent pathway, were internalised in a manner independent from endosomal acidification, and required routing through the Golgi apparatus, as reported for modeccin and volkensin. Stenodactylin showed greater uptake, exocytosis and re-uptake of non-degraded RIP than lanceolin and volkensin, whereas volkensin had the highest residual activity after being released from the cell. Conclusions The high cytotoxicity of RIPs from the Adenia genus may depend on the following: high affinity binding to the cell and efficient endocytosis, intracellular routing that appears similar to that of other ricin-like toxic RIPs, partial resistance to proteolysis, and, regarding stenodactylin, high accumulation in cell. General significance The data provide a model that could lead to new strategies for anti-cancer therapy and neuroscience studies.

Compound C sensitizes Caki renal cancer cells to TRAIL-induced apoptosis through reactive oxygen species-mediated down-regulation of c-FLIP L and Mcl-1

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anticancer drugs, is considered as a new strategy for anticancer therapy. Compound C, a cell-permeable pyrrazolopyrimidine derivative, acts as a potent, selective, reversible ATP-competitive inhibitor of AMP-activated protein kinase (AMPK). In this study, we show that compound C sensitizes Caki human renal cancer cells, but not normal human skin fibroblast cells (HSF) and human mesangial cells, to TRAIL-mediated apoptosis. However, AMPK siRNA failed to affect TRAIL-mediated apoptosis in Caki cells and transduction of dominant negative AMPK rather attenuated TRAIL-induced apoptosis, indicating that the effect of compound C on sensitization of TRAIL-induced apoptosis is independent of AMPK activity. Interestingly, we found that down-regulation of c-FLIP L and Mcl-1 contributes to compound C-enhanced TRAIL-induced apoptosis. Reduced expression of c-FLIP L and Mcl-1 were caused by the decreased protein stability of c-FLIP L and Mcl-1, but not by their transcriptional control, in compound C-treated cells. Pretreatment with N-acetyl- l-cysteine (NAC) significantly inhibited the cell death induced by the combined treatment with compound C and TRAIL as well as recovered the expression levels of c-FLIP L and Mcl-1 down-regulated by the combinatory treatment with compound C plus TRAIL, suggesting that compound C-stimulated TRAIL-induced apoptosis appears to be dependent on the generation of reactive oxygen species for down-regulation of c-FLIP L and Mcl-1. Taken together, the present study demonstrates that compound C enhances TRAIL-induced apoptosis in human renal cancer cells by ROS-mediated c-FLIP L and Mcl-1 down-regulation.