looking-for-driver-pathways-acquired-resistance-to-targeted-therapy

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS

Despite the low efficacy of conventional antitumour drugs, chemotherapy remains an essential tool in controlling advanced gastric and oesophageal cancers. We aimed to provide a biological rationale based on the sorafenib-taxotere interaction for the clinical treatment of gastric cancer. In vitro experiments were performed on four human gastric cancer cell lines (GK2, AKG, KKP and NCI-N87). Cytotoxicity was evaluated by sulforhodamine B (SRB) assay, cell cycle perturbations, apoptosis and mitotic catastrophe were assessed by flow cytometric and microscopic analyses, and protein expression was studied by Western blot. In the in vivo experiments, nude mice xenografted with the most resistant line were treated with sorafenib and docetaxel singly or in association. Sorafenib inhibited cell growth (IG(50) values ranged from 3.4 to 8.1 μM) and caused down-regulation of MAP-K/ERK phosphorylation and of mcl-1 and p-bad expression after a 48-hr exposure. Apoptosis induction was associated with caspase-3 and -9 activation and mitochondrial membrane depolarization. The drug combination enhanced apoptosis (up to 80%) and produced a synergistic interaction when low doses of the taxane preceded administration of the antityrosine kinase. This synergism was probably due to the induction of an anomalous multidiploid G0-G1 peak and to consequent mitotic catastrophe, which increased sensitivity to sorafenib. Consistent with in vitro results, the docetaxel-sorafenib sequence exhibited high therapeutic efficacy in NCI-N87 mouse xenografts producing tumour weight inhibition (> 65%), tumour growth delay (up to 25 days) and increased mouse survival (30%). Our findings suggest the potential clinical usefulness of treatment with sorafenib and docetaxel for advanced gastric cancer.

Low-dose taxotere enhances the ability of sorafenib to induce apoptosis in gastric cancer models.

Lung cancer is the leading cause of cancer mortality worldwide and despite efforts made to improve clinical results, continuing poor survival rates indicate that novel therapeutic approaches are needed. Valproic acid (VPA), a short-chain branched fatty acid used mainly for the treatment of epilepsy and bipolar disorder, has been shown to inhibit class I histone deacetylases (HDAC-I), a group of enzymes involved in chromatin remodeling and which are thought to play a role in tumor development. Although evidence of VPA's therapeutic efficacy has also been observed in patients with solid tumors, the very high concentration required to induce antitumor activity limits its clinical usefulness. We used a panel of NSCLC cell lines to evaluate the activity and mechanisms of action of organosulfur valproic acid derivatives, a promising new class of compounds designed to improve the safety and efficacy of the valproic acid molecule and created by coupling it with a hydrogen sulfide (H(2) S)-releasing moiety. Our results highlighted the increased cytotoxic activity of the novel organosulfur derivatives, ACS33 and ACS2, with respect to VPA, starting from low concentrations. In particular, ACS2 exhibited important pro-apoptotic activity triggered by the mitochondrial pathway and also showed anti-invasion potential. Furthermore, our in vitro results identified a highly effective combination schedule of ACS2 and cisplatin capable of inducing a synergistic interaction even when the two drugs were used at low concentrations, which could prove a valid alternative to traditional chemotherapeutic regimens used for advanced lung cancer. Further studies are needed to confirm these preliminary findings.

Organosulfur derivatives of the HDAC inhibitor valproic acid sensitize human lung cancer cell lines to apoptosis and to cisplatin cytotoxicity.

The management of hormone-refractory prostate cancer represents a major challenge in the therapy of this tumor, and identification of novel androgen receptor antagonists is needed to render treatment more effective. We analyzed the activity of two novel androgen receptor antagonists, (S)-11 and (R)-9, in in vitro and in vivo experimental models of hormone-sensitive or castration-resistant prostate cancer (CRPC). In vitro experiments were performed on LNCaP, LNCaP-AR, LNCaP-Rbic and VCaP human prostate cancer cells. Cytotoxic activity was assessed by SRB and BrdU uptake, AR transactivation by luciferase reporter assay and PSA levels by Real Time RT-PCR and ELISA assays. Cell cycle progression-related markers were evaluated by western blot. In vivo experiments were performed on SCID mice xenografted with cells with different sensitivity to hormonal treatment. In hormone-sensitive LNCaP and LNCaP-AR cells, the latter expressing high androgen receptor levels, (R)-9 and (S)-11 exhibited a higher cytotoxic effect compared to that of the reference compound ((R)-bicalutamide), also in the presence of the synthetic androgen R1881. Furthermore, the cytotoxic effect produced by (R)-9 was higher than that of (S)-11 in the two hormone-resistant LNCaP-AR and VCaP cells. A significant reduction in PSA levels was observed after exposure to both molecules. Moreover, (S)-11 and (R)-9 inhibited DNA synthesis by blocking the androgen-induced increase in cyclin D1 protein levels. In vivo studies on the toxicological profile of (R)-9 did not reveal the presence of adverse events. Furthermore, (R)-9 inhibited tumor growth in various in vivo models, especially LNCaP-Rbic xenografts, representative of recurrent disease. Our in vitro results highlight the antitumor activity of the two novel molecules (R)-9 and (S)-11, making them a potentially attractive option for the treatment of CRPC.

Effect of small molecules modulating androgen receptor (SARMs) in human prostate cancer models.

Although two-dimensional (2-D) monolayer cell cultures provide important information on basic tumor biology and radiobiology, they are not representative of the complexity of three-dimensional (3-D) solid tumors. In particular, new models reproducing clinical conditions as closely as possible are needed for radiobiological studies to provide information that can be translated from bench to bedside.

In vitro irradiation system for radiobiological experiments.

Sufficient knowledge regarding cellular and molecular basis of lung cancer progression and metastasis would help in the development of novel and effective strategies for the treatment of lung cancer. 4HPR is a synthetic retinoid with potential anti-tumor activity but is still limited because of its poor bioavailability. The use of albumin as a complexing agent for a hydrophobic drug is expected to improve the water solubility and consequently their bioavailability.This study investigated the antitumor activity of a novel complex between albumin and 4-HPR in a mouse model of human lung cancer and focuses on role and mechanism of Cav-1 mainly involved in regulating cancer and ACSVL3 mainly connected with tumor growth. Their expressions were assayed by immunohistochemistry and qRT-PCR, to demonstrate the reduction of the tumor growth following the drug treatment. Our results showed a high antitumor activity of 4HPR-HSA by reduction of the volume of tumor mass and the presence of a high level of apoptotic cell by TUNEL assay. The downregulation of Cav-1 and ACSVL3 suggested a reduction of tumor growth. In conclusion, we demonstrated the great potential of 4HPR-HSA in the treatment of lung cancer. More data about the mechanism of drug delivery the 4HPR-HSA are necessary.

Anti-tumor activity of fenretinide complexed with human serum albumin in lung cancer xenograft mouse model.

Cell counting is one of the basic needs of most biological experiments. Numerous methods and systems have been studied to improve the reliability of counting. However, at present, manual cell counting performed with a hemocytometer still represents the gold standard, despite several problems limiting reproducibility and repeatability of the counts and, at the end, jeopardizing their reliability in general. We present our own approach based on image processing techniques to improve counting reliability. It works in two stages: first building a high-resolution image of the hemocytometer's grid, then counting the live and dead cells by tagging the image with flags of different colours. In particular, we introduce GridMos (http://sourceforge.net/p/gridmos), a fully-automated mosaicing method to obtain a mosaic representing the whole hemocytometer's grid. In addition to offering more significant statistics, the mosaic "freezes" the culture status, thus permitting analysis by more than one operator. Finally, the mosaic achieved can thus be tagged by using an image editor, thus markedly improving counting reliability. The experiments performed confirm the improvements brought about by the proposed counting approach in terms of both reproducibility and repeatability, also suggesting the use of a mosaic of an entire hemocytometer's grid, then labelled trough an image editor, as the best likely candidate for the new gold standard method in cell counting.

Improving reliability of live/dead cell counting through automated image mosaicing.

Volume is one of the most important features for the characterization of a tumour on a macroscopic scale. It is often used to assess the effectiveness of care treatments, thus making its correct evaluation a crucial issue for patient care. Similarly, volume is a key feature on a microscopic scale. Multicellular cancer spheroids are 3D tumour models widely employed in pre-clinical studies to test the effects of drugs and radiotherapy treatments. Very few methods have been proposed to estimate the tumour volume arising from a 2D projection of multicellular spheroids, and even fewer have been designed to provide a 3D reconstruction of the tumour shape. In this work, we propose Reconstruction and Visualization from a Single Projection (ReViSP), an automatic method conceived to reconstruct the 3D surface and estimate the volume of single cancer multicellular spheroids, or even of spheroid cultures. As the input parameter ReViSP requires only one 2D projection, which could be a widefield microscope image. We assessed the effectiveness of our method by comparing it with other approaches. To this purpose, we used a new strategy that allowed us to achieve accurate volume measurements based on the analysis of home-made 3D objects, built by mimicking the spheroid morphology. The results confirmed the effectiveness of our method for both 3D reconstruction and volume assessment. ReViSP software is distributed as an open source tool.

Cancer multicellular spheroids: volume assessment from a single 2D projection.

The potential of a spheroid tumor model composed of cells in different proliferative and metabolic states for the development of new anticancer strategies has been amply demonstrated. However, there is little or no information in the literature on the problems of reproducibility of data originating from experiments using 3D models. Our analyses, carried out using a novel open source software capable of performing an automatic image analysis of 3D tumor colonies, showed that a number of morphology parameters affect the response of large spheroids to treatment. In particular, we found that both spheroid volume and shape may be a source of variability. We also compared some commercially available viability assays specifically designed for 3D models. In conclusion, our data indicate the need for a pre-selection of tumor spheroids of homogeneous volume and shape to reduce data variability to a minimum before use in a cytotoxicity test. In addition, we identified and validated a cytotoxicity test capable of providing meaningful data on the damage induced in large tumor spheroids of up to diameter in 650 μm by different kinds of treatments.

3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained.

HER2-positive advanced gastric cancer patients frequently develop resistance to trastuzumab through mechanisms still poorly understood. In breast cancer, other members of the HER-family are known to be involved in trastuzumab-resistance, as is overexpression of the scaffold protein IQGAP1. In the present work, we investigated acquired resistance to trastuzumab in gastric cancer experimental models. Trastuzumab-resistant (HR) subclones derived from 3 HER2-overexpressing gastric cancer cells were generated and characterized for alterations in HER2-signaling mechanisms by next-generation sequencing, immunohistochemical, western blot and qRT-PCR techniques, and molecular modeling analysis. All subclones showed a reduced growth rate with respect to parental cell lines but each had a different resistance mechanism. In NCI N87 HR cells, characterized by a marked increase in HER2-signaling pathways with respect to the parental cell line, trastuzumab sensitivity was restored when IQGAP1 expression was silenced. AKG HR subclone showed higher HER3 protein expression than the parental line. High nuclear HER4 levels were observed in KKP HR cells. In conclusion, our study revealed that high IQGAP1 expression leads to resistance to trastuzumab in gastric cancer. Furthermore, 2 new mutations of the HER2 gene that may be involved in acquired resistance were identified in AKG HR and KKP HR subclones.

Preclinical evidence of multiple mechanisms underlying trastuzumab resistance in gastric cancer.

We investigated the correlation between ploidy or S-phase fraction (SPF) and the clinical pathological characteristics of patients with peritoneal carcinomatosis from ovarian cancer. We also assessed their relation with the in vivo and in vitro response to several chemotherapeutic agents.

DNA ploidy and S-phase fraction analysis in peritoneal carcinomatosis from ovarian cancer: correlation with clinical pathological factors and response to chemotherapy.

Article	Year
Low-dose taxotere enhances the ability of sorafenib to induce apoptosis in gastric cancer models. Journal of cellular and molecular medicine\| PubMed ID: 20015197	2011
Organosulfur derivatives of the HDAC inhibitor valproic acid sensitize human lung cancer cell lines to apoptosis and to cisplatin cytotoxicity. Journal of cellular physiology\| PubMed ID: 22212895	2012
Effect of small molecules modulating androgen receptor (SARMs) in human prostate cancer models. PloS one\| PubMed ID: 23667504	2013
In vitro irradiation system for radiobiological experiments. Radiation oncology (London, England)\| PubMed ID: 24180359	2013
Anti-tumor activity of fenretinide complexed with human serum albumin in lung cancer xenograft mouse model. Oncotarget\| PubMed ID: 25015569	2014
Improving reliability of live/dead cell counting through automated image mosaicing. Computer methods and programs in biomedicine\| PubMed ID: 25438936	2014
Cancer multicellular spheroids: volume assessment from a single 2D projection. Computer methods and programs in biomedicine\| PubMed ID: 25561413	2015
3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Scientific reports\| PubMed ID: 26752500	2016
Preclinical evidence of multiple mechanisms underlying trastuzumab resistance in gastric cancer. Oncotarget\| PubMed ID: 26919099	2016
DNA ploidy and S-phase fraction analysis in peritoneal carcinomatosis from ovarian cancer: correlation with clinical pathological factors and response to chemotherapy. OncoTargets and therapy\| PubMed ID: 29033584	2017

Anna Tesei

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