Endocytosis In Cancer Cells Research Articles

Abstract PO3-24-02: Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers

Abstract Introduction: Current FDA-approved immunotherapeutics for triple-negative breast cancer are only available for a subset of patients, highlighting the need to develop improved immune-targeting strategies. VISTA (V-domain Ig suppressor of T cell activation) is a single-pass transmembrane immune checkpoint receptor that is an emerging clinical target for cancer immunotherapy. It is expressed widely on lymphoid and myeloid cells in healthy individuals, is commonly expressed on cells of the tumor microenvironment and can be abnormally upregulated on tumor cells in a wide variety of tumors. Although VISTA confers quiescence to naïve T cells, molecular mechanisms responsible for this effect remain unclear. Also, the effects of VISTA expression on tumor cells remains poorly defined. Methods: Multi-omic profiling was performed in human breast cancer cells lines to determine immune regulatory genes controlled by growth factor stimulation. Human triple-negative breast cancer tumor specimens (N=248) were examined by immunohistochemistry for expression of VISTA, PDL1, PD1 and FoxP3. VISTA expression was engineered into human and mouse triple-negative breast cancer cell lines and growth metrics were characterized in vitro and in immunocompetent (syngeneic) and immunodeficient (xenograft) mouse tumor models. Membrane receptor trafficking and localization of EGFR was performed in human TNBC cells lines expressing VISTA and VISTA mutants. Proximity biotinylation identified protein partners that interacted with VISTA’s intracellular domain. Deletion mutagenesis was performed to identify motifs required for biochemical interactions between VISTA and clathrin adaptor proteins. Clathrin adapter localization was examined in WT and VISTA knock-out cell lines. Cytokine response, clathrin adaptor localization and tumor growth kinetics were measured in TNBC cell lines expressing VISTA or VISTA mutants. Results: A class of human triple-negative breast cancers were identified with high VISTA expression, low tumor infiltrating lymphocytes and low proliferative index (8-20% of all TNBCs). Tumor models demonstrated that enforced expression of VISTA in tumor cells caused growth suppression, even in the absence of an immune system. This effect was mapped to the cytoplasmic domain of VISTA, which lacks defined signaling domains. Through biochemical approaches, the clathrin-adaptor molecules NUMB and GULP1 were found in complex with a VISTA intracellular NPGF motif. This motif sequestered NUMB at early endosomes, resulting in diminished NUMB recycling for EGFR endocytosis, thereby causing growth suppression. VISTA impaired NUMB recycling by blocking Rab11 effector complex formation. VISTA NPGF mutations did not disrupt cytokine secretion in mixed lymphocyte assays. VISTA-targeted antibodies did not disrupt NUMB sequestration. VISTA+ tumors were selectively sensitive to VISTA-blocking antibodies. Conclusions: VISTA+ TNBC have distinct features including slower proliferative index and increased sensitivity to VISTA-blocking antibodies. This work identifies mechanisms by which VISTA enforces a quiescent cellular state by sequestering clathrin adapter proteins away from sites of endocytosis in cancer cells. This is the first description of intracellular molecular mechanisms controlled by VISTA’s intracellular tail. It may inform features of the VISTA-dependent immune checkpoint that could be exploited to improve anti-tumor immunity. Citation Format: Yan Zhao, Fatima Charles, Andrew Lipchik, Yan Peng, James Ford, Melinda Telli, Song Zhang, Allison Kurian, Michael Snyder, Joshua Gruber. Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-02.

Abstract B027: Molecular characterization and therapeutic approaches for VISTA+ triple-negative breast cancers

Abstract Introduction: VISTA (V-domain Ig suppressor of T cell activation) is a single-pass transmembrane immune checkpoint receptor that is an emerging clinical target for cancer immunotherapy. Although VISTA confers quiescence to naïve T cells, molecular mechanisms responsible for this effect remain unclear. Also, the effects of VISTA expression on tumor cells remains poorly defined. Methods: Multi-omic profiling was performed in human breast cancer cells lines to determine immune regulatory genes. Human triple-negative breast cancers (N=248) were examined by immunohistochemistry for expression of VISTA, PDL1, PD1 and FoxP3. VISTA expression was engineered into human and mouse triple-negative breast cancer cell lines and growth was characterized in vitro and in mouse models. Membrane receptor trafficking and localization of EGFR and clathrin adapters was performed in cells expressing VISTA, VISTA mutants and VISTA knockouts. Proximity biotinylation identified protein partners that interacted with VISTA’s intracellular domain. Deletion mutagenesis was performed to identify functional VISTA cytoplasmic motifs. Cytokine response and tumor growth kinetics were measured in TNBC mouse models expressing VISTA or VISTA mutants. Results: A class of human triple-negative breast cancers were identified with high VISTA expression, low tumor infiltrating lymphocytes and low proliferative index (8-20% of all TNBCs). Tumor models demonstrated that enforced expression of VISTA in tumor cells caused growth suppression, even in the absence of an immune system. This effect was mapped to the cytoplasmic domain of VISTA, which lacks defined signaling domains. Through biochemical approaches, the clathrin-adaptor molecules NUMB and GULP1 were found in complex with a VISTA intracellular NPGF motif. This motif sequestered NUMB at early endosomes, resulting in diminished NUMB recycling for EGFR endocytosis, thereby causing growth suppression. VISTA impaired NUMB recycling by blocking Rab11 effector complex formation. VISTA NPGF mutations did not disrupt cytokine secretion in mixed lymphocyte assays. VISTA-targeted antibodies did not disrupt NUMB sequestration. VISTA+ tumors were selectively sensitive to VISTA-blocking antibodies. Conclusions: VISTA+ TNBC have distinct features including slower proliferative index and increased sensitivity to VISTA-blocking antibodies. This work identifies mechanisms by which VISTA enforces a quiescent cellular state by sequestering clathrin adapter proteins away from sites of endocytosis in cancer cells. This is the first description of intracellular molecular mechanisms controlled by VISTA’s intracellular tail. It may inform features of the VISTA-dependent immune checkpoint that could be exploited to improve anti-tumor immunity. Integration of these findings with ongoing VISTA clinical trials will be discussed. Citation Format: Joshua Gruber, Yan Zhao, Fatima Charles, Priyanka Reddy, Kristina Paul, Virginia Ezenwa, Marisa Juntilla, Idit Sagiv-Barfi, Yan Peng, Song Zhang, Allison Kurian, Michael Snyder. Molecular characterization and therapeutic approaches for VISTA+ triple-negative breast cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B027.

Development of Targeted EGFR Degradation for Cancer Treatment

Chemotherapeutic interruption of signaling pathways at receptor tyrosine kinases is an important strategy for attenuating cancer progression. We have synthesized a new generation of polyfunctionalized heterocyclic compounds that bind to an allosteric site in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). The bound compounds induce degradation of the receptor by endocytosis in cancer cells. The Hsp90α chaperone binds to EGFR and is significantly lost during endocytosis, thereby contributing to the reduction of client proteins. Induced EGFR depletion leads to inactivation of downstream signaling due to sequestration of the Bim sensor protein for cytoskeletal proteins, provoking cell detachment from the extracellular matrix and ultimately cancer cell death. The role of glutamine in maintaining the phosphorylation status of EGFR-mediated signaling pathways can be postulated as «no glutamine, no EGFR signaling». Targeted degradation of EGFR is attractive for aiming to attenuate metastatic progression and to override the drug resistance of malignant tumors.

1-Deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells

Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids (SLs) that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I, resulting in the synthesis and accumulation of 1-deoxysphingolipids (deoxySLs). These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine levels can promote 1-deoxySL synthesis, they impact numerous other metabolic pathways important for cancer cells. Here, we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxySL toxicity in cancer cells. We determined that both alanine treatment and SPTLC1C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, we found that spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxySL synthesis was induced via SPTLC1C133W expression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxySL synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.

Medicated Scaffolds Prepared with Hydroxyapatite/Streptomycin Nanoparticles Encapsulated into Polylactide Microfibers.

The preparation, characterization, and controlled release of hydroxyapatite (HAp) nanoparticles loaded with streptomycin (STR) was studied. These nanoparticles are highly appropriate for the treatment of bacterial infections and are also promising for the treatment of cancer cells. The analyses involved scanning electron microscopy, dynamic light scattering (DLS) and Z-potential measurements, as well as infrared spectroscopy and X-ray diffraction. Both amorphous (ACP) and crystalline (cHAp) hydroxyapatite nanoparticles were considered since they differ in their release behavior (faster and slower for amorphous and crystalline particles, respectively). The encapsulated nanoparticles were finally incorporated into biodegradable and biocompatible polylactide (PLA) scaffolds. The STR load was carried out following different pathways during the synthesis/precipitation of the nanoparticles (i.e., nucleation steps) and also by simple adsorption once the nanoparticles were formed. The loaded nanoparticles were biocompatible according to the study of the cytotoxicity of extracts using different cell lines. FTIR microspectroscopy was also employed to evaluate the cytotoxic effect on cancer cell lines of nanoparticles internalized by endocytosis. The results were promising when amorphous nanoparticles were employed. The nanoparticles loaded with STR increased their size and changed their superficial negative charge to positive. The nanoparticles’ crystallinity decreased, with the consequence that their crystal sizes reduced, when STR was incorporated into their structure. STR maintained its antibacterial activity, although it was reduced during the adsorption into the nanoparticles formed. The STR release was faster from the amorphous ACP nanoparticles and slower from the crystalline cHAp nanoparticles. However, in both cases, the STR release was slower when incorporated in calcium and phosphate during the synthesis. The biocompatibility of these nanoparticles was assayed by two approximations. When extracts from the nanoparticles were evaluated in cultures of cell lines, no cytotoxic damage was observed at concentrations of less than 10 mg/mL. This demonstrated their biocompatibility. Another experiment using FTIR microspectroscopy evaluated the cytotoxic effect of nanoparticles internalized by endocytosis in cancer cells. The results demonstrated slight damage to the biomacromolecules when the cells were treated with ACP nanoparticles. Both ACP and cHAp nanoparticles were efficiently encapsulated in PLA electrospun matrices, providing functionality and bioactive properties.

Mechanism of Inhibition of Hydroxyapatite Nanoparticles on Cancer Cells and Recent Advances in the Field

Background: Calcium phosphates are chemically similar to bone minerals. The biocompatibility, bioactivity, and high similarity of these substances to body organs such as bone have made them a good choice for disease diagnosis and treatment. Here, the main use of calcium phosphates is diagnosis and treatment of cancers. Hydroxyapatite is a bioactive material with a high affinity for DNA and protein. Recently, hydroxyapatite nanoparticles exhibit different properties than those of bulk hydroxyapatite in chemistry and biology. In general, the anticancer effects of hydroxyapatite nanoparticles have been attributed to high amounts of endocytosis in cancer cells and inhibits of protein synthesis in cells. Methods: Herein, we evaluated the structure, properties, and methods of synthesis of hydroxyapatite nanoparticles. Moreover, the mechanism of inhibition of hydroxyapatite nanoparticles on cancer cells and recent advances in this field have been examined. Conclusion: Hydroxyapatite nanoparticles had the ability to eliminate the development of cancer cells in vitro and in vivo. In the live tissue environment, injection of hydroxyapatite nanoparticles at the tumor’s surrounding area had a significant decrease in tumor size (about 50%).

Abstract 1215: Targeting TIP1 with a novel radiosensitizing antibody-drug conjugate (ADC) enhances the therapeutic efficacy of radiation for non-small cell lung carcinoma treatment

Abstract Tax-interacting protein 1 (TIP1) is a multifunctional protein involved in cellular motility, cellular adhesion, migration, cancer cell protection from ionizing radiation, and metastasis. We found that TIP1 is a radiation inducible antigen that is translocated to the cancer cell surface. Antibody-drug-conjugates (ADCs) are emerging as promising anti-cancer immunoconjugates. ADCs carry antitumor payloads and allow site-specific targeting of cancer with increased cytotoxicity and decreased side effect profiles as compared to conventional chemotherapy. Monomethyl auristatin E (MMAE) is a potent radiosensitizing microtubule inhibitor with enhanced cancer cytotoxicity in combination with radiotherapy. We developed ADCs against radiation-inducible TIP1 for lung cancer treatment in combination with radiation. Out of the various anti-TIP1 antibodies, we prioritized the lead antibody that shows high affinity to TIP1 protein on the cancer cell surface and in vivo irradiated lung cancers. Our lead antibody, 7H5 undergoes endocytosis in cancer cells in vitro. We conjugated 7H5 to VcMMAE. Hydrophobic interaction chromatography (HIC) was performed to determine drug-to-antibody ratio (DAR). We obtained an average DAR of 3.7. Cytotoxic potential of 7H5-VcMMAE was evaluated in A549 and H1299 NSCLC cells. Cells were exposed to 7H5-VcMMAE or 7H5-antibody alone at various doses. At 10nM concentration, ~70% reduction in viable cells was observed following 7H5-VcMMAE treatment compared to 7H5 alone in both A549 (p=0.0032) and H1299 cells (p=0.0010). 7H5-VcMMAE sensitized A549 and H1299 cells to radiation as observed by significantly decreased (p<0.001) surviving fraction compared to 7H5 alone. In vivo therapeutic efficacy of the 7H5-VcMMAE was determined using mice bearing hind-limb human NSCLC tumors. The ADC showed a prolonged delay in tumor growth and improved tumor control was achieved with IR + 7H5-VcMMAE in A549 and H1299 hind-limb tumor models. Furthermore, mice in all groups displayed no toxicity associated with treatment. Our study suggests that targeting radiation-inducible TIP1 with a radiosensitizing ADC is a promising strategy to enhance therapeutic efficacy in lung cancer. This novel approach of targeting cancer using ADCs to radiation-inducible antigens will lead to clinical trials in lung cancer patients treated with radiotherapy. Citation Format: Calvin Lewis, Abhay K. Singh, Dinesh Thotala, Dennis E. Hallahan, Vaishali Kapoor. Targeting TIP1 with a novel radiosensitizing antibody-drug conjugate (ADC) enhances the therapeutic efficacy of radiation for non-small cell lung carcinoma treatment [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 1215.

Targeting a Radiosensitizing Antibody-Drug Conjugate to a Radiation-Inducible Antigen.

We recently discovered that anti-TIP1 antibody activates endocytosis in cancer cells, which facilitates retention of antibody and dissociation of a conjugated drug. To improve the pharmacokinetics and cancer specificity of radiosensitizing drugs, we utilized antibody-drug conjugates (ADCs) that bind specifically to radiation-inducible antigen, TIP1, on non-small cell lung cancer (NSCLC). This approach exploits the long circulation time of antibodies to deliver a radiosensitizing drug to cancer each day during radiotherapy. Antibodies to TIP1 were prioritized based on affinity, cancer-specific binding, and internalization. The lead antibody, 7H5, was conjugated with a cytotoxic drug MMAE because of its ability to radiosensitize cancer. Cytotoxicity, colony formation, and tumor growth studies were performed with 7H5-VcMMAE in combination with radiation. 7H5 showed a high affinity to recombinant TIP1 protein and radiation-inducible TIP1 on the cancer cell surface. 7H5 undergoes endocytosis in NSCLC cells in vitro. We obtained an average drug-to-antibody ratio (DAR) of 4.25 for 7H5-VcMMAE. A 70% reduction in viable cells was observed following 7H5-VcMMAE treatment compared with 7H5 alone in both A549 and H1299 cells. 7H5-VcMMAE sensitized NSCLC cells to radiation, thereby significantly decreasing the surviving fraction. The ADC combined with radiation showed a prolonged delay in tumor growth and improved survival in A549 and H1299 tumor models. Targeting radiation-inducible TIP1 with a radiosensitizing ADC is a promising strategy to enhance the therapeutic efficacy of NSCLC. This novel approach of targeting with ADCs to radiation-inducible antigens will lead to clinical trials in lung cancer patients treated with radiotherapy.

Supramolecular nanoparticles self-assembled from reduction-responsive cabazitaxel prodrugs for effective cancer therapy.

Using hydrophobic cabazitaxel as a target anticancer drug, we show that the conjugation of oligo(ethylene glycol)-oligolactide (OEG-OLA) via a self-immolative linkage induces the self-assembly of the resulting prodrug into injectable nanoparticles. The nanoparticles release chemically unmodified cabazitaxel after endocytosis in cancer cells. With the optimal conjugate, the nanotherapy not only potently induces tumor regression but also has a higher safety margin in animals than the free drug administered in its clinical formulation. Our studies highlight the design rationale that attaching a short amphiphilic oligomer to a toxic drug can convert it to a self-deliverable and safe nanotherapy.

Assessing Cobalt Metal Nanoparticles Uptake by Cancer Cells Using Live Raman Spectroscopy.

PurposeNanotechnology applied to cancer treatment is a growing area of research in nanomedicine with magnetic nanoparticle-mediated anti-cancer drug delivery systems offering least possible side effects. To that end, both structural and chemical properties of commercial cobalt metal nanoparticles were studied using label-free confocal Raman spectroscopy.Materials and MethodsCrystal structure and morphology of cobalt nanoparticles were studied by XRD and TEM. Magnetic properties were studied with SQUID and PPMS. Confocal Raman microscopy has high spatial resolution and compositional sensitivity. It, therefore, serves as a label-free tool to trace nanoparticles within cells and investigate the interaction between coating-free cobalt metal nanoparticles and cancer cells. The toxicity of cobalt nanoparticles against human cells was assessed by MTT assay.ResultsSuperparamagnetic Co metal nanoparticle uptake by MCF7 and HCT116 cancer cells and DPSC mesenchymal stem cells was investigated by confocal Raman microscopy. The Raman nanoparticle signature also allowed accurate detection of the nanoparticle within the cell without labelling. A rapid uptake of the cobalt nanoparticles followed by rapid apoptosis was observed. Their low cytotoxicity, assessed by means of MTT assay against human embryonic kidney (HEK) cells, makes them promising candidates for the development of targeted therapies. Moreover, under a laser irradiation of 20mW with a wavelength of 532nm, it is possible to bring about local heating leading to combustion of the cobalt metal nanoparticles within cells, whereupon opening new routes for cancer phototherapy.ConclusionLabel-free confocal Raman spectroscopy enables accurately localizing the Co metal nanoparticles in cellular environments. The interaction between the surfactant-free cobalt metal nanoparticles and cancer cells was investigated. The facile endocytosis in cancer cells shows that these nanoparticles have potential in engendering their apoptosis. This preliminary study demonstrates the feasibility and relevance of cobalt nanomaterials for applications in nanomedicine such as phototherapy, hyperthermia or stem cell delivery.

ESR Method in Monitoring of Nanoparticle Endocytosis in Cancer Cells.

Magnetic nanoparticles are extensively studied for their use in diagnostics and medical therapy. The behavior of nanoparticles after adding them to cell culture is an essential factor (i.e., whether they attach to a cell membrane or penetrate the membrane and enter into the cell). The present studies aimed to demonstrate the application of electron spin resonance (ESR) as a suitable technique for monitoring of nanoparticles entering into cells during the endocytosis process. The model nanoparticles were composed of magnetite iron (II, III) oxide core functionalized with organic unit containing nitroxide radical 4-hydroxy-TEMPO (TEMPOL). The research studies included breast cancer cells, as well as model yeast and human microvascular endothelial cells. The results confirmed that the ESR method is suitable for studying the endocytosis process of nanoparticles in the selected cells. It also allows for direct monitoring of radical cellular processes.

Targeted Graphene Oxide Networks: Cytotoxicity and Synergy with Anticancer Agents.

An effective strategy to inhibit endocytosis in cancer cells is presented where modified net-type graphene oxide (GO) sheets, bound with multiple cell surface receptors, are introduced and synthesized as novel anticancer agents. The results suggest that the binding connects GO sheets with neighboring lipid rafts, neutralizes endocytosis, and causes metabolic deprivation. As a result, tumor cell survival and proliferation are reduced. Live cell confocal microscopy imaging reveals that GO-PEGFA (folate-PEGylated GO) (PEG, polyethylene glycol) is internalized by tumor cells, while GO-PEGRGD (tripeptide Arg-Gly-Asp PEGylated GO) associates with the external cell membrane (not internalized). In vitro exposure of tumor cells to GO-PEGFA or GO-PEGRGD reduces the cell viability by 35%, compared to 50% reduction using methotrexate (100 μM). The combination of modified GO sheets with methotrexate or doxorubicin shows a greater toxicity (80% reduction in cell viability) than the individual agents. The proposed setup demonstrates a significant synergy in limiting tumor cell growth.

Endocytic Selective Toxicity of Rhodamine 6G nanoGUMBOS in Breast Cancer Cells.

Herein, we report on the role of endocytosis in the selective chemotherpeutic toxicity of rhodamine 6G (R6G) based nanomaterials, i.e., nanoGUMBOS, that are derived from a group of uniform materials based on organic salts (GUMBOS). Evaluation of cellular uptake in the presence and absence of endocytosis inhibitors suggests nanoGUMBOS internalization via clathrin-mediated endocytosis in cancer cells and reveals lack of endocytic internalization in normal cells. Results from characterization of these nanomaterials suggest that endocytic internalization in cancer cells leads to nanoGUMBOS dissociation within the endosomal environment. This ultimately results in selective cytotoxicity of the nanoGUMBOS for cancer cells with no toxicity toward normal cells under examined conditions. Following examination of the selectivity mechanism, in vivo investigations were performed to examine potential therapeutic properties of these nanoparticles. Remarkably, nanoGUMBOS treatment using a mouse xenograft model reduced the tumor volume by 50% suggesting retention of in vitro therapeutic properties in vivo. These results corroborate the selective behavior of nanoGUMBOS and demonstrate their in vivo therapeutic effects, providing further insight into the possible use of these nanomaterials as potential chemotherapeutic agents.

Abstract 966: Oncogenic signaling pathways differentially regulate clathrin-mediated endocytosis in cancer cells

Abstract Clathrin-mediated endocytosis (CME), the major endocytic pathway, regulates the rates of internalization of receptors as well as their downstream signaling activities. Recently, our lab has discovered that signaling can reciprocally regulate CME in cancer cells. The crosstalk between signaling and CME contributes to abnormal trafficking of signaling receptors and altered downstream signaling, leading to enhanced cancer metastasis. Accordingly, it is important to understand the nature and extent of interactions between CME and intracellular signaling in cancer cell biology. To address this issue, we targeted a set of prioritized kinases that are often implicated in cancer-relevant signaling pathways using validated pharmacologic inhibitors and compared their effects on CME function using different models for human noncancerous and cancer cells. We found that inhibition of several kinases selectively affected CME function in cancer cells. Notably, ERK inhibition had the most significant and consistent effects on CME activity across different types of cancer cells, while not affecting CME in several noncancerous cell lines tested. We established that a second ERK inhibitor, and importantly inhibition of the essential upstream kinase MEK, had the same cancer cell-specific effects on CME activity. Furthermore, we found that inhibition of ERK dramatically reduced the rate of clathrin-coated pit (CCP) initiation selectively in cancer cells. Characterization of one potential ERK substrate, FCHSD2, showed that knockdown of FCHSD2 had the similar cancer cell-specific effects on CME activity and on CCP initiation rate and that the cells depleted for FCHSD2 were insensitive to ERK inhibition. Surprisingly, the expression level of FCHSD2 is positively correlated with higher cancer patient survival rate, suggesting that FCHSD2 might be a tumor suppressor regulated by ERK. Our study provides new insight into the mechanisms and consequences of the reciprocal regulation of signaling and CME in cancer cells. Citation Format: Guan-Yu Xiao, Sandra Schmid. Oncogenic signaling pathways differentially regulate clathrin-mediated endocytosis in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 966.

Biotin-Tagged Polysaccharide Vesicular Nanocarriers for Receptor-Mediated Anticancer Drug Delivery in Cancer Cells.

Biotin-conjugated multistimuli-responsive polysaccharide vesicular nanocarriers are designed and developed, for the first time, to accomplish receptor-mediated endocytosis in cancer cells and to deliver anticancer drugs to intracellular compartments. For this purpose, a new renewable hydrophobic unit was custom designed with redox-degradable disulfide and enzyme-biodegradable aliphatic ester chemical linkages, and it was conjugated along with biotin on the dextran backbone. The dextran derivative self-assembled into nanovesicles of <200 nm in size, which were characterized by dynamic and static light scattering, electron, and atomic force microscopes. Avidin-HABA assay established the high affinity of biotin-tagged dextran vesicles toward membrane-receptors up to 25 nM concentration. Doxorubicin-hydrochloride (DOX.HCl)-loaded dextran vesicles exhibited stable formulation in phosphate-buffered saline (PBS) and fetal bovine serum (FBS). Redox-degradation by glutathione (GSH) showed 60% drug release, whereas lysosomal esterase enzyme enabled >98% drug release in 12 h. Confocal microscope and flow cytometry-assisted time-dependent cellular uptake studies revealed that the biotin-receptors overexpressed in cervical cancer cells (HeLa) exhibited larger drug accumulation through the receptor-assisted endocytosis process. This process enabled the delivery of higher amount of DOX and significantly enhanced the killing in cancer cells (HeLa) compared to wild-type mouse embryonic fibroblast cells (WT-MEF, normal cells). Control experiments such as biotin pretreatment in cancer cells and energy-suppressed cellular uptake at 4 °C further supported the occurrence of receptor-mediated endocytosis by the biotin-tagged polymer vesicles. This report provides first insights into the targeted polysaccharide vesicle platform, and the proof-of-concept is successfully demonstrated in biotin receptor-overexpressed cervical cancer cells.

SNARE Protein in Cellular Membrane Trafficking, Its Regulation and as a Potential Target for Cancer Treatment

The role of SNARE [soluble NSF (N-ethylmaleimide-sensitive factor) accessory protein receptor] protein in cellular trafficking, membrane fusion and vesicle release in synaptic nerve terminals is described. The purpose of this review is to highlight the role of SNAREs in vital inflammatory conditions in maturing dendritic cells in order to retain the capacity to present new antigens together with altered cytokine secretory functions. This role of SNAREs can be used as novel targets for therapy in inflammatory diseases. The essential mechanism of SNAREs proteins for regulation of tumour formation through multiple signals and transportation pathways is also discussed. Finally, this review summarizes the current knowledge of SNARE proteins in regulating endocytosis in cancer cells and the possible therapeutic applications related to the pathogenesis of tumor formation.

Oseltamivir-conjugated polymeric micelles prepared by RAFT living radical polymerization as a new active tumor targeting drug delivery platform.

Targeted drug delivery using polymeric nanostructures has been at the forefront of cancer research, engineered for safer, more efficient and effective use of chemotherapy. Here, we designed a new polymeric micelle delivery system for active tumor targeting followed by micelle-drug internalization via receptor-induced endocytosis. We recently reported that oseltamivir phosphate targets and inhibits Neu1 sialidase activity associated with receptor tyrosine kinases such as epidermal growth factor receptors (EGFRs) which are overexpressed in cancer cells. By decorating micelles with oseltamivir, we investigated whether they actively targeted human pancreatic PANC1 cancer cells. Amphiphilic block copolymers with oseltamivir conjugated at the hydrophilic end, oseltamivir-pPEGMEMA-b-pMMA (oseltamivir-poly(polyethylene glycol methyl ether methacrylate)-block-poly(methyl methacrylate), were synthesized using reversible addition-fragmentation chain transfer (RAFT) living radical polymerization. Oseltamivir-conjugated micelles have self-assembling properties to give worm-like micellar structures with molecular weight of 80 000 g mol(-1). Oseltamivir-conjugated water soluble pPEGMEMA, dose dependently, both inhibited sialidase activity associated with Neu1, and reduced viability of PANC1 cells. In addition, oseltamivir-conjugated micelles, labelled with a hydrophobic fluorescent dye within the micelle core, were subsequently internalized by PANC1 cells. Blocking cell surface Neu1 with anti-Neu1 antibody, reduced internalization of oseltamivir-conjugated micelles, demonstrating that Neu1 binding linked to sialidase inhibition were prerequisite steps for subsequent internalization of the micelles. The mechanism of internalization is likely that of receptor-induced endocytosis demonstrating potential as a new nanocarrier system for not only targeting a tumor cell, but also for directly reducing viability through Neu1 inhibition, followed by intracellular delivery of hydrophobic cytotoxic chemotherapeutics.

Different inhibitory effect and mechanism of hydroxyapatite nanoparticles on normal cells and cancer cells in vitro and in vivo.

Hydroxyapatite (HAP), similar to inorganic phase in bones, shows good biocompatibility and bioactivity as bone defect repairing material. Recently, nanoscaled HAP shows the special properties differing from bulk HAP in physics, chemistry and biology. This paper demonstrates that HAP nanoparticle (nHAP) possesses the ability for inhibiting cancer cell growth in vitro and in vivo. In vitro, after treatment with nHAP for 3 days, proliferation of human cancer cells are inhibited by more than 65% and by less than 30% for human normal cells. In vivo, injection of nHAP in transplanted tumor results in significant reduction (about 50%) of tumor size. The anticancer effect of nHAP is mainly attributed to high amount by endocytosis in cancer cells and inhibition on protein synthesis in cells. The abundant nHAP internalized in cancer cells around endoplasmic reticulum may inhibit the protein synthesis by decreasing the binding of mRNA to ribosome due to its high adsorption capacity for ribosome and arrest cell cycle in G0/G1 phase. nHAP shows no ROS-involved cytotoxicity and low cytotoxicity to normal cells. These results strongly suggest that nHAP can inhibit cancer cell proliferation and have a potential application in cancer treatment.

Selective cellular uptake and induction of apoptosis of cancer-targeted selenium nanoparticles

Selenium nanoparticles (SeNPs) have garnered a great deal of attention as potential cancer therapeutic payloads. However, the invivo targeting drug delivery has been challenging. Herein, we describe the synthesis of tansferrin (Tf)-conjugated SeNPs and its use as a cancer-targeted drug delivery system to achieve enhanced cellular uptake and anticancer efficacy. Tf as targeting ligand significantly enhances the cellular uptake of doxorubicin (DOX)-loaded SeNPs through clathrin-mediated and caveolae/lipid raft-mediated endocytosis in cancer cells overexpressing transferrin receptor, and increases their selectivity between cancer and normal cells. DOX-loaded and Tf-conjugated SeNPs (Tf-SeNPs) exhibits unprecedented enhanced cytotoxicity toward cancer cells through induction of apoptosis with the involvement of intrinsic and extrinsic pathways. Internalized Tf-SeNPs triggers intracellular ROS overproduction, thus activates p53 and MAPKs pathways to promote cell apoptosis. In the nude mice xenograft experiment, Tf-SeNPs significantly inhibits the tumor growth via induction of p53-mediated apoptosis. This cancer-targeted design of SeNPs opens a new path for synergistic treating of cancer with higher efficacy and decreased side effects.

Pegylated siRNA-loaded calcium phosphate nanoparticle-driven amplification of cancer cell internalization in vivo

The cell membrane is a critical barrier to effective delivery for many therapeutics, including those which are nanoparticle-based. Improving nanoparticle transport across the cell membrane remains a fundamental challenge. Cancer cells preferentially internalized pegylated calcium phosphate nanoparticles over normal epithelial cells. Furthermore, non-cytotoxic levels of doxorubicin markedly amplified this difference by increasing free unbound caveolin-1 and resulted in enhanced caveolin-mediated nanoparticle endocytosis in cancer cells. Engineered pegylated siRNA-loaded triple-shell calcium phosphate nanoconstructs incorporating ultra-low levels of doxorubicin recapitulated these effects and delivered increased numbers of siRNA into cancer cells with target-specific results. Systemic administration of nanoparticles in vivo demonstrated highly preferential entry into tumors, little bystander organ biodistribution, and significant tumor growth arrest. In conclusion, siRNA-loaded calcium phosphate nanoparticles incorporating non-cytotoxic amounts of doxorubicin markedly enhances nanoparticle internalization and results in increased payload delivery with concomitant on-target effects.