Altered sphingolipid metabolism in N-(4-hydroxyphenyl) retinamide resistant A2780 human ovarian carcinoma cells

In the present work, we studied the effects of fenretinide (N-(4-hydroxyphenyl)retinamide (HPR)), a hydroxyphenyl derivative of all-trans-retinoic acid, on sphingolipid metabolism and expression in human ovarian carcinoma A2780 cells. A2780 cells, which are sensitive to a pharmacologically achievable HPR concentration, become 10-fold more resistant after exposure to increasing HPR concentrations. Our results showed that HPR was able to induce a dose- and time-dependent increase in cellular ceramide levels in sensitive but not in resistant cells. This form of resistance in A2780 cells was not accompanied by the overexpression of multidrug resistance-specific proteins MDR1 P-glycoprotein and multidrug resistance-associated protein, whose mRNA levels did not differ in sensitive and resistant A2780 cells. HPR-resistant cells were characterized by an overall altered sphingolipid metabolism. The overall content in glycosphingolipids was similar in both cell types, but the expression of specific glycosphingolipids was different. Specifically, our findings indicated that glucosylceramide levels were similar in sensitive and resistant cells, but resistant cells were characterized by a 6-fold lower expression of lactosylceramide levels and by a 6-fold higher expression of ganglioside levels than sensitive cells. The main gangliosides from resistant A2780 cells were identified as GM3 and GM2. The possible metabolic mechanisms leading to this difference were investigated. Interestingly, the mRNA levels of glucosylceramide and lactosylceramide synthases were similar in sensitive and resistant cells, whereas GM3 synthase mRNA level and GM3 synthase activity were remarkably higher in resistant cells

Circulating tumor DNA and disease recurrence in early stage breast cancer: From a case-control study to a prospective longitudinal trial

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4-oxo-N-(4-hydroxyphenyl)retinamide: Two Independent Ways to Kill Cancer Cells

BACKGROUND: The retinoid 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) is a polar metabolite of fenretinide (4-HPR) very effective in killing cancer cells of different histotypes, able to inhibit 4-HPR-resistant cell growth and to act synergistically in combination with the parent drug. Unlike 4-HPR and other retinoids, 4-oxo-4-HPR inhibits tubulin polymerization, leading to multipolar spindle formation and mitotic arrest. Here we investigated whether 4-oxo-4-HPR, like 4-HPR, triggered cell death also via reactive oxygen species (ROS) generation and whether its antimicrotubule activity was related to a ROS-dependent mechanism in ovarian (A2780), breast (T47D), cervical (HeLa) and neuroblastoma (SK-N-BE) cancer cell lines. METHODOLOGY/PRINCIPAL FINDINGS: We provided evidence that 4-oxo-4-HPR, besides acting as an antimicrotubule agent, induced apoptosis through a signaling cascade starting from ROS generation and involving endoplasmic reticulum (ER) stress response, Jun N-terminal Kinase (JNK) activation, and upregulation of the proapoptotic PLAcental Bone morphogenetic protein (PLAB). Through time-course analysis and inhibition of the ROS-related signaling pathway (upstream by vitamin C and downstream by PLAB silencing), we demonstrated that the antimitotic activity of 4-oxo-4-HPR was independent from the oxidative stress induced by the retinoid. In fact, ROS generation occurred earlier than mitotic arrest (within 30 minutes and 2 hours, respectively) and abrogation of the ROS-related signaling pathway did not prevent the 4-oxo-4-HPR-induced mitotic arrest. CONCLUSIONS/SIGNIFICANCE: These data indicate that 4-oxo-4-HPR anticancer activity is due to at least two independent mechanisms and provide an explanation of the ability of 4-oxo-4-HPR to be more potent than the parent drug and to be effective also in 4-HPR-resistant cell lines. In addition, the double mechanism of action could allow 4-oxo-4-HPR to efficiently target tumour and to eventually counteract the development of drug resistance

Challenges in Using Circulating miRNAs as Cancer Biomarkers

In the last years, circulating miRNAs have emerged as a new class of promising cancer biomarkers. Independent studies have shown the feasibility of using these small RNAs as tools for the diagnosis and prognosis of different types of malignancies as well as for predicting and possibly monitoring treatment response. However, despite an initial enthusiasm for their possible clinical application, widespread inconsistencies have been observed among the studies, and miRNA-based tools still represent the object of research within clinical diagnostic or treatment protocols. The poor overlap of results could be explained, at least in part, by preanalytical and analytical variables and donor-related factors that could generate artefacts, impairing an accurate quantification of circulating miRNAs. In fact, critical issues are represented by nonuniform sample choice, handling, and processing, as well as by blood cell contamination in sample preparation and lack of consensus for data normalization. In this review, we address the potential technical biases and individual-related parameters that can influence circulating miRNA studies’ outcome. The exciting potential of circulating miRNAs as cancer biomarkers could confer an important advance in the disease management, but their clinical significance might not be proven without a global consensus of procedures and standardized protocols for their accurate detection

AF1q: A Novel Mediator of Basal and 4-HPR-Induced Apoptosis in Ovarian Cancer Cells

<div><h3>Background</h3><p>Fenretinide (4-HPR) is a synthetic retinoid that exhibits potent antitumor and chemopreventive activities against different malignancies, including ovarian tumors. We previously showed that in ovarian cancer cells, 4-HPR induces apoptosis through a signaling cascade starting from reactive oxygen species (ROS) generation and involving endoplasmic reticulum (ER) stress response, Jun N-terminal Kinase (JNK) activation, and induction of the proapoptotic PLAcental Bone morphogenetic protein (PLAB). Since recent studies have shown that the oncogene ALL1-fused from chromosome 1q (AF1q), a retinoic acid target gene, is implicated in apoptosis induction by several therapeutic agents, we investigated its possible involvement in the apoptosis induced by 4-HPR in ovarian cancer cells.</p> <h3>Methodology/Principal Findings</h3><p>Protein expression analysis, performed in ovarian cancer cells and extended to other histotypes (breast, neuroblastoma, and cervical), revealed that 4-HPR enhanced AF1q expression in cancer cells sensitive to the retinoid but not in resistant cells. Through gene silencing, AF1q was found functionally involved in 4-HPR-induced apoptosis in A2780, an ovarian cancer cell line highly sensitive to retinoid growth inhibitory and apoptotic effects. Inhibition of the signaling intermediates of the 4-HPR apoptotic cascade showed that AF1q upregulation was depended on prior generation of ROS, induction of ER stress response, JNK activation, and PLAB upmodulation. Finally, we found that direct overexpression of AF1q, in the absence of external stimuli, increased apoptosis in ovarian cancer cell lines.</p> <h3>Conclusions/Significance</h3><p>The study expands the knowledge of the 4-HPR mechanism of action, which has not yet been completely elucidated, identifying AF1q as a novel mediator of retinoid anticancer activity. In addition, we demonstrate, for the first time, that AF1q plays a role in the onset of basal apoptosis in ovarian cancer cells, thus providing new information about the activity of this protein whose biologic functions are mostly unknown.</p> </div

Relationship between AF1q upregulation and 4-HPR-induced signaling cascade.

<p>Western blot analysis for AF1q expression in A2780 cells treated for 24 hours with 5 µM 4-HPR, with or without 100 µM vitamin C (A), 10 µM salubrinal (B), or 10 µM SP600125 (C). (D) Western blot analysis for AF1q expression in A2780 cells stably transfected with a plasmid containing a PLAB siRNA or a scrambled nonsilencing siRNA following addition of 5 µM 4-HPR for 24 hours. As a control for loading, the blots were incubated with actin antibody.</p

Early Modulation of Circulating MicroRNAs Levels in HER2-Positive Breast Cancer Patients Treated with Trastuzumab-Based Neoadjuvant Therapy

Càncer de mama; MicroRNA circulants; TrastuzumabCáncer de mama; MicroARN circulantes; TrastuzumabBreast cancer; Circulating microRNAs; TrastuzumabCirculating microRNA (ct-miRNAs) are able to identify patients with differential response to HER2-targeted therapy. However, their dynamics are largely unknown. We assessed 752 miRNAs from 52 NeoALTTO patients with plasma pairs prior and two weeks after trastuzumab. Increased levels of ct-miR-148a-3p and ct-miR-374a-5p were significantly associated with pathological complete response (pCR) (p = 0.008 and 0.048, respectively). At a threshold ≥ the upper limit of the 95%CI of the mean difference, pCR resulted 45% (95%CI 24%–68%), and 44% (95%CI 22%–69%) for ct-miR-148a-3p and ct-miR-374a-5p, respectively. Notably, ct-miR-148a-3p retained its predictive value (OR 3.42, 95%CI 1.23–9.46, p = 0.018) in bivariate analysis along with estrogen receptor status. Combined information from ct-miR-148a-3p and ct-miR140-5p, which we previously reported to identify trastuzumab-responsive patients, resulted in greater predictive capability over each other, with pCR of 54% (95%CI 25%–81%) and 0% (95%CI 0%–31%) in ct-miR-148a/ct-miR-140-5p high/present and low/absent, respectively. GO and KEGG analyses showed common enriched terms between the targets of these ct-miRNAs, including cell metabolism regulation, AMPK and MAPK signaling, and HCC progression. In conclusion, early modulated ct-miR-148-3p may inform on the functional processes underlying treatment response, integrate the information from already available predictive biomarkers, and identify patients likely to respond to single agent trastuzumab-based neoadjuvant therapy.The NeoALTTO study was sponsored by GlaxoSmithKline; Lapatinib is an asset of Novartis AG as of 2 March 2015. This substudy was sponsored by the Italian Association for Cancer Research Foundation (Fondazione AIRC) through the MFAG 14361 and IG 20774 to SDC, and the IG 16900 to MGD, and by the Italian Ministry of Health through funds obtained by an Italian law that allows taxpayers to allocate the “5 × 1000” share of their payments to research

Effect of AF1q overexpression in the onset of basal apoptosis.

<p>(A) Immunofluorescence analysis of A2780 cells transiently transfected with GFP alone (upper panels) or GFP-tagged AF1q vector (AF1q-GFP) (lower panels). After 48 hours, GFP and AF1q-GFP cells were stained with Hoechst 33342 and nuclear morphology was examined with a fluorescent microscope. Cells of interest are marked by arrows. Cells with condensed and fragmented nuclei were identified and scored as apoptotic cells. One experiment representative of three is shown. The scale bar represents 10 µm. (B) Apoptosis was represented as percentage of apoptotic cells per 100 green fluorescent cells (at least 200 cells per sample) in A2780 (left panel) and OVCAR-3 (right panel) cells transfected as in (A). Data represent the mean±S.D. of three independent experiments. Asterisks indicate significant difference (P<0.05).</p