Activation of autophagy following [HuArgI (Co)-PEG5000]-induced arginine deprivation mediates cell death in colon cancer cells.

Deregulating cellular energetics by reprogramming metabolic pathways, including arginine metabolism, is critical for cancer cell onset and survival. Drugs that target the specific metabolic requirements of cancer cells have emerged as promising targeted cancer therapeutics. In this study, we investigate the therapeutic potential of targeting colon cancer cells using arginine deprivation induced by a pegylated cobalt-substituted recombinant human Arginase I [HuArgI (Co)-PEG5000]. Four colon cancer cell lines were tested for their sensitivity to [HuArgI (Co)-PEG5000] as well as for their mechanism of cell death following arginine deprivation. All four cell lines were sensitive to arginine deprivation induced by [HuArgI (Co)-PEG5000]. All cells expressed ASS1 and were rescued from arginine deprivation-induced cytotoxicity by the addition of excess L-citrulline, indicating they are partially auxotrophic for arginine. Mechanistically, cells treated with [HuArgI (Co)-PEG5000] were negative for AnnexinV and lacked caspase activation. Further investigation revealed that arginine deprivation leads to a marked and prolonged activation of autophagy in both Caco-2 and T84 cell lines. Finally, we show that [HuArgI (Co)-PEG5000] causes cell death by sustained activation of autophagy as evidenced by the decrease in cell cytotoxicity upon treatment with chloroquine, an autophagy inhibitor. Altogether, these data demonstrate that colon cancer cells are partially auxotrophic for arginine and sensitive to [HuArgI (Co)-PEG5000]-induced arginine deprivation. They also show that the activation of autophagy does not play protective roles but rather, induces cytotoxicity and leads to cell death.

Tolerant/Persister Cancer Cells and the Path to Resistance to Targeted Therapy.

The capacity of cancer to adapt to treatment and evolve is a major limitation for targeted therapies. While the role of new acquired mutations is well-established, recent findings indicate that resistance can also arise from subpopulations of tolerant/persister cells that survive in the presence of the treatment. Different processes contribute to the emergence of these cells, including pathway rebound through the release of negative feedback loops, transcriptional rewiring mediated by chromatin remodeling and autocrine/paracrine communication among tumor cells and within the tumor microenvironment. In this review, we discuss the non-genetic mechanisms that eventually result in cancer resistance to targeted therapies, with a special focus on those involving changes in gene expression.

ZZW-115-dependent inhibition of NUPR1 nuclear translocation sensitizes cancer cells to genotoxic agents.

Establishing the interactome of the cancer-associated stress protein Nuclear Protein 1 (NUPR1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair, and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1, thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further report that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system, indicating that NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation-dependent functions of key proteins involved in the DDR.

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations.

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

PML hyposumoylation is responsible for the resistance of pancreatic cancer.

The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is mainly due to its rapidly acquired resistance to all conventional treatments. Despite drug-specific mechanisms of resistance, none explains how these cells resist the stress induced by any kind of anticancer treatment. Activation of stress-response pathways relies on the post-translational modifications (PTMs) of involved proteins. Among all PTMs, those mediated by the ubiquitin family of proteins play a central role. Our aim was to identify alterations of ubiquitination, neddylation, and sumoylation associated with the multiresistant phenotype and demonstrate their implications in the survival of PDAC cells undergoing treatment. This approach pointed at an alteration of promyelocytic leukemia (PML) protein sumoylation associated with both gemcitabine and oxaliplatin resistance. We could show that this alteration of PML sumoylation is part of a general mechanism of drug resistance, which in addition involves the abnormal activation of NF-κB and cAMP response element binding pathways. Importantly, using patient-derived tumors and cell lines, we identified a correlation between the levels of PML expression and sumoylation and the sensitivity of tumors to anticancer treatments.-Swayden, M., Alzeeb, G., Masoud, R., Berthois, Y., Audebert, S., Camoin, L., Hannouche, L., Vachon, H., Gayet, O., Bigonnet, M., Roques, J., Silvy, F., Carrier, A., Dusetti, N., Iovanna, J. L., Soubeyran, P. PML hyposumoylation is responsible for the resistance of pancreatic cancer.

Can the plasma PD-1 levels predict the presence and efficiency of tumor-infiltrating lymphocytes in patients with metastatic melanoma?

BACKGROUND: The immune response in melanoma patients is locally affected by presence of tumor-infiltrating lymphocytes (TILs), generally divided into brisk, nonbrisk, and absent. Several studies have shown that a greater presence of TILs, especially brisk, in primary melanoma is associated with a better prognosis and higher survival rate. PATIENTS AND METHODS: We investigated by enzyme-linked immunosorbent assay (ELISA) the correlation between PD-1 levels in plasma and the presence/absence of TILs in 28 patients with metastatic melanoma. RESULTS: Low plasma PD-1 levels were correlated with brisk TILs in primary melanoma, whereas intermediate values correlated with the nonbrisk TILs, and high PD-1 levels with absent TILs. Although the low number of samples did not allow us to obtain a statistically significant correlation between the plasma PD-1 levels and the patients' overall survival depending on the absence/presence of TILs, the median survival of patients having brisk type TILs was 5 months higher than that of patients with absent and nonbrisk TILs. CONCLUSIONS: This work highlights the ability of measuring the plasma PD-1 levels in order to predict the prognosis of patients with untreated metastatic melanoma without a BRAF mutation at the time of diagnosis.

Upcoming Revolutionary Paths in Preclinical Modeling of Pancreatic Adenocarcinoma.

To date, PDAC remains the cancer having the worst prognosis with mortality rates constantly on the rise. Efficient cures are still absent, despite all attempts to understand the aggressive physiopathology underlying this disease. A major stumbling block is the outdated preclinical modeling strategies applied in assessing effectiveness of novel anticancer therapeutics. Current in vitro preclinical models have a low fidelity to mimic the exact architectural and functional complexity of PDAC tumor found in human set, due to the lack of major components such as immune system and tumor microenvironment with its associated chemical and mechanical signals. The existing PDAC preclinical platforms are still far from being reliable and trustworthy to guarantee the success of a drug in clinical trials. Therefore, there is an urgent demand to innovate novel in vitro preclinical models that mirrors with precision tumor-microenvironment interface, pressure of immune system, and molecular and morphological aspects of the PDAC normally experienced within the living organ. This review outlines the traditional preclinical models of PDAC namely 2D cell lines, genetically engineered mice, and xenografts, and describing the present famous approach of 3D organoids. We offer a detailed narration of the pros and cons of each model system. Finally, we suggest the incorporation of two off-center newly born techniques named 3D bio-printing and organs-on-chip and discuss the potentials of swine models and in silico tools, as powerful new tools able to transform PDAC preclinical modeling to a whole new level and open new gates in personalized medicine.

Pancreatic cancer chemo-resistance is driven by tumor phenotype rather than tumor genotype.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest forms of cancer. A major reason for this situation is the fact that these tumors are already resistant or become rapidly resistant to all conventional therapies. Like any transformation process, initiation and development of PDCA are driven by a well known panel of genetic alterations, few of them are shared with most cancers, but many mutations are specific to PDAC and are partially responsible for the great inter-tumor heterogeneity. Importantly, this knowledge has been inefficient in predicting response to anticancer therapy, or in establishing diagnosis and prognosis. Hence, the pre-existing or rapidly acquired resistance of pancreatic cancer cells to therapeutic drugs rely on other parameters and features developed by the cells and/or the micro-environment, that are independent of their genetic profiles. This review sheds light on all major phenotypic, non genetic, alterations known to play important roles in PDAC cells resistance to treatments and therapeutic escape.

Chloroquine plays a cell-dependent role in the response to treatment of pancreatic adenocarcinoma.

In this study, our aim is to assess the role played by autophagy and its inhibition in the different PDAC cellular compartments, and its involvement in chemo-resistance using primary human pancreatic cancer-derived cells (PCC) and Cancer Associated Fibroblasts (CAF). Autophagy flux, as measured by LC3-I and -II in the presence of Chloroquine, showed a variable level in PCC and CAFs. We found no correlation between autophagy level and degree of tumor differentiation. Association of Chloroquine with gemcitabine, 5FU, oxaliplatin, irinotecan and docetaxel revealed that its effect on survival is cell- and drug-dependent in vitro and in vivo. In addition, we demonstrated that autophagy in CAFs can play an important role in sensitizing PDAC to anticancer treatments since its inhibition increased the resistance of PCCs to gemcitabine. In conclusion, this work clearly shows a heterogeneity in the effect of Chloroquine and highlights a role of CAFs autophagy in sensitizing tumors to treatments. It also reveals that the role of autophagy is more complex than expected in PDAC as well as its sensitivity to treatments.