D. Massihnia

Stabilizing versus destabilizing the microtubules: A double-edge sword for an effective cancer treatment option?

Microtubules are dynamic and structural cellular components involved in several cell functions, including cell shape, motility, and intracellular trafficking. In proliferating cells, they are essential components in the division process through the formation of the mitotic spindle. As a result of these functions, tubulin and microtubules are targets for anticancer agents. Microtubule-targeting agents can be divided into two groups: microtubule-stabilizing, and microtubule-destabilizing agents. The former bind to the tubulin polymer and stabilize microtubules, while the latter bind to the tubulin dimers and destabilize microtubules. Alteration of tubulin-microtubule equilibrium determines the disruption of the mitotic spindle, halting the cell cycle at the metaphase-anaphase transition and, eventually, resulting in cell death. Clinical application of earlier microtubule inhibitors, however, unfortunately showed several limits, such as neurological and bone marrow toxicity and the emergence of drug-resistant tumor cells. Here we review several natural and synthetic microtubule-targeting agents, which showed antitumor activity and increased efficacy in comparison to traditional drugs in various preclinical and clinical studies. Cryptophycins, combretastatins, ombrabulin, soblidotin, D-24851, epothilones and discodermolide were used in clinical trials. Some of them showed antiangiogenic and antivascular activity and others showed the ability to overcome multidrug resistance, supporting their possible use in chemotherapy.

Triple negative breast cancer: shedding light onto the role of pi3k/akt/mtor pathway

Breast cancer is one of the most widespread carcinoma and one of the main causes of cancer-related death worldwide, especially in women aged between 35 and 75 years. Among the different subtypes, triple negative breast cancer (TNBC) is characterized by the total absence of the estrogen-receptor (ER) and progesteron-receptor (PR) expression as well as the lack of human epidermal growth factor receptor 2 (HER2) overexpression or gene amplification. These biological characteristics confer to TNBC a higher aggressiveness and relapse risk along with poorer prognosis compared to other subtypes. Indeed, 5-years survival rate is still low and almost all patients die, despite any adjuvant treatment which at moment represents the heading pharmacological approach. To date, several clinical trials have been designed to investigate the potential role of some molecular markers, such as VEGF, EGFR, Src and mTOR, for targeted treatments in TNBC. In fact, many inhibitors of the PI3K/AKT/mTOR pathway, frequently de-regulated in TNBC, are acquiring a growing interest and several inhibitors are in preclinical development or already in early phase clinical trials. In this Review, we investigated the role of the PI3K/AKT/mTOR pathway in TNBC patients, by summarizing the molecular features that led to the distinction of different histotypes of TNBC. Furthermore, we provided an overview of the inhibition mechanisms of the mTOR and PI3K/AKT signaling pathways, highlighting the importance of integrating biological and clinical data for the development of mTOR inhibitors in order to implement targeted therapies for TNBC patients.

Phospho-Akt overexpression is prognostic and can be used to tailor the synergistic interaction of Akt inhibitors with gemcitabine in pancreatic cancer

BackgroundThere is increasing evidence of a constitutive activation of Akt in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and chemoresistance. Therefore, we evaluated the expression of phospho-Akt in PDAC tissues and cells, and investigated molecular mechanisms influencing the therapeutic potential of Akt inhibition in combination with gemcitabine.MethodsPhospho-Akt expression was evaluated by immunohistochemistry in tissue microarrays (TMAs) with specimens tissue from radically-resected patients (n = 100). Data were analyzed by Fisher and log-rank test. In vitro studies were performed in 14 PDAC cells, including seven primary cultures, characterized for their Akt1 mRNA and phospho-Akt/Akt levels by quantitative-RT-PCR and immunocytochemistry. Growth inhibitory effects of Akt inhibitors and gemcitabine were evaluated by SRB assay, whereas modulation of Akt and phospho-Akt was investigated by Western blotting and ELISA. Cell cycle perturbation, apoptosis-induction, and anti-migratory behaviors were studied by flow cytometry, AnnexinV, membrane potential, and migration assay, while pharmacological interaction with gemcitabine was determined with combination index (CI) method.ResultsImmunohistochemistry of TMAs revealed a correlation between phospho-Akt expression and worse outcome, particularly in patients with the highest phospho-Akt levels, who had significantly shorter overall and progression-free-survival. Similar expression levels were detected in LPC028 primary cells, while LPC006 were characterized by low phospho-Akt. Remarkably, Akt inhibitors reduced cancer cell growth in monolayers and spheroids and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028, while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a reduced expression of ribonucleotide reductase, potentially facilitating gemcitabine cytotoxicity. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. This combination significantly increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. However, targeting the key glucose transporter Glut1 resulted in similar apoptosis induction in LPC006 cells.ConclusionsThese data support the analysis of phospho-Akt expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies.

Dietary restriction: could it be considered as speed bump on tumor progression road?

Dietary restrictions, including fasting (or long-term starvation), calorie restriction (CR), and short-term starvation (STS), are considered a strong rationale that may protect against various diseases, including age-related diseases and cancer. Among dietary approaches, STS, in which food is not consumed during designed fasting periods but is typically not restricted during designated feeding periods, seems to be more suitable, because other dietary regimens involving prolonged fasting periods could worsen the health conditions of cancer patients, being they already naturally prone to weight loss. Until now, the limited amount of available data does not point to a single gene, pathway, or molecular mechanism underlying the benefits to the different dietary approaches. It is well known that the healthy effect is mediated in part by the reduction of nutrient-related pathways. The calorie restriction and starvation (long- and short-term) also suppress the inflammatory response reducing the expression, for example, of IL-10 and TNF-α, mitigating pro-inflammatory gene expression and increasing anti-inflammatory gene expression. The dietary restriction may regulate both genes involved in cellular proliferation and factors associated to apoptosis in normal and cancer cells. Finally, dietary restriction is an important tool that may influence the response to chemotherapy in preclinical models. However, further data are needed to correlate dietary approaches with chemotherapeutic treatments in human models. The aim of this review is to discuss the effects of various dietary approaches on the cancer progression and therapy response, mainly in preclinical models, describing some signaling pathways involved in these processes.

A headlight on liquid biopsies: a challenging tool for breast cancer management

Breast cancer is the most frequent carcinoma and second most common cause of cancer-related mortality in postmenopausal women. The acquisition of somatic mutations represents the main mechanism through which cancer cells overcome physiological cellular signaling pathways (e.g., PI3K/Akt/mTOR, PTEN, TP53). To date, diagnosis and metastasis monitoring is mainly carried out through tissue biopsy and/or re-biopsy, a very invasive procedure limited only to certain locations and not always feasible in clinical practice. In order to improve disease monitoring over time and to avoid painful procedure such as tissue biopsy, liquid biopsy may represent a new precious tool. Indeed, it represents a basin of “new generation” biomarkers that are spread into the bloodstream from both primary and metastatic sites. Moreover, elevated concentrations of circulating tumor DNA (ctDNA) as well as circulating tumor cells (CTCs) have been found in blood plasma of patients with various tumor types. Nowadays, several new approaches have been introduced for the detection and characterization of CTCs and ctDNA, allowing a real-time monitoring of tumor evolution. This review is focused on the clinical relevance of liquid biopsy in breast cancer and will provide an update concerning CTCs and ctDNA utility as a tool for breast cancer patient monitoring during the course of disease.

Liquid Biopsy in Esophageal, Gastric, and Pancreatic Cancers

Esophageal, gastric and pancreatic cancers represent lethal solid tumors and endoscopic biopsies are the main tool for the histological characterization of these cancer. However, there are many problems for tissue biopsy, together with possible surgical complications, tumor diffusion, and incorrect and/or negative results. Furthermore, in many cases there is no sufficient material from both primary tumors and metastasis for a proper molecular characterization. For these reasons, liquid biopsies represent an attractive minimally invasive methodology for the management of oncological patients. Liquid biopsy includes circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating microRNAs and proteins and extracellular vesicles. Nevertheless, CTCs and ctDNA are the most commonly studied components of liquid biopsy and may acquire an important role also in esophageal, gastric and pancreatic cancers for a better desease management. In this chapter we will discuss how CTCs, exosomes and circulating nucleic acids can be used to obtain prognostic and predictive information and also could be a useful tool for early diagnosis.

Liquid Biopsy in Breast Cancer

Breast cancer (BC) to date remains the most common cancer in women. Nowadays, BC is often diagnosed at local disease stage, and, after surgery, based on individual’s risk of relapse, the patients undergo adjuvant systemic treatment to decrease the risk of recurrence. Current BC classification and assessment remain strongly based on clinicopathological criteria, including patient age, tumor size, lymph node invasion, histological type, and grade. According to standard practice, the choice of treatment strategy includes assays for estrogen (ER) and progesterone (PgR) receptor expression levels, overexpression of human epidermal growth factor receptor 2 (Her-2), or amplification status of the correlate oncogene, but also histological grade and Ki67 to evaluate proliferation of tumor cells. Nevertheless, the established clinicopathological parameters are not sufficient anymore for clinical decision-making and should be combined with genomic profiling to estimate recurrence risk and identify high-risk BC patients (prognostic value) and predict optimal treatment for each disease subgroup (predictive value). The information obtained from standard tumor tissue sampling cannot be the same for the whole tumor and offer a static picture of disease. The constant molecular change of tumor cell population, spatial and temporal, requires a noninvasive approach, for real-time picture of disease. Liquid biopsy is a useful tool to follow the continuously evolving genomic landscape of breast cancer.

Abstract 1855: Role of mTOR inhibition in triple-negative breast cancer

BACKGROUND: Triple-negative breast cancers (TNBC) represent the 10-17% of all diagnosed breast cancers (BC) and are characterized by the absence of ER/PgR expression, HER2 amplification and often show a basal-like phenotype. TNBC are often diagnosed in patients with BRCA1 germline mutation and unfortunately treatment options are still limited. The mTOR (Mammalian Target Of Rapamycin) pathway seems to play an important role in BC pathogenesis and it is possible to target this pathway by inhibitors such as rapamycin. In human BC cross talk between ER/PgR receptors signaling and the mTOR pathway is believed to be responsible for resistance to hormone therapy probably due to a down regulation of hormone receptors. Based on these evidences we have hypothesized that the inhibitors of mTOR pathway may lead to the up-regulation of ER, PgR and HER2 in TNBC cell lines. METHODS: For this study we used TNBC cells (MDA-MB-231 and BT20) cultured in DMEM:F12 (Dulbecco9s Modified Eagle9s Medium) with 10% bovine serum (FBS), 100 U/mL penicillin (1%) and 50 mg/mL of streptomycin under standard conditions (37°C in an atmosphere composed of 16% O2, 79% N2 and 5% CO2). Cells were initially treated with Rapamycin (1, 5, 10, 15 microM) for 24, 48, and 72h in order to verify if the drug determines a blockade of cell proliferation. Before drug administration, the cells were subjected to serum starvation by eliminating serum from culture medium. The evaluation of cell viability following the administration of the drug was carried out using the MTT assay. Real time PCR analyses were carried out in order to evaluate gene expression modifications of ER, PgR and HER2 receptors, through Taqman probe chemistry. RESULTS: The preliminary cell viability experiments conducted on different TNBC cell lines (MDA-MB-231 and BT20), showed no significant cytotoxic effects by increasing Rapamycin concentrations (1, 5 and 10 microM) after 72h treatment, except for the higher concentration (15 microM) for which a cytotoxic effect was observed. The following qPCR approach highlighted significant variations in estrogen and progesterone receptor gene expression for the TNBC cell lines after 24, 48, 72h with 1, 5, 10 microM Rapamycin. In particular their expression level resulted up-regulated. Unlikely no association between mTOR inhibition and HER2 expression level were identified, suggesting no effects of mTOR inhibition on HER2 expression. CONCLUSION AND FUTURE PERSPECTIVES: To these preliminary results suggest that the mTOR inhibition leads to re-expression of hormone receptors. This finding supports a potential clinical application of mTOR inhibition in TNBC. The perspective of phenotype change upon rapamycin treatment prompts new therapeutic scenarios. However, further investigations are needed to explain the biological mechanisms driving these changes. Citation Format: Daniela Massihnia, Giuseppe Bronte, Marta Castiglia, Nadia Barraco, Antonina Cangemi, Alessandro Perez, Daniele Fanale, Gianni Pantuso, Salvatore Vieni, Valentina Calo, Christian D. Rolfo, Viviana Bazan, Antonio Russo. Role of mTOR inhibition in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1855.