Anna Tessari

miR-9 and miR-200 regulate PDGFRβ-mediated endothelial differentiation of tumor cells in triple-negative breast cancer

Organization of cancer cells into endothelial-like cell-lined structures to support neovascularization and to fuel solid tumors is a hallmark of progression and poor outcome. In triple-negative breast cancer (TNBC), PDGFRβ has been identified as a key player of this process and is considered a promising target for breast cancer therapy. Thus, we aimed at investigating the role of miRNAs as a therapeutic approach to inhibit PDGFRβ-mediated vasculogenic properties of TNBC, focusing on miR-9 and miR-200. In MDA-MB-231 and MDA-MB-157 TNBC cell lines, miR-9 and miR-200 promoted and inhibited, respectively, the formation of vascular-like structures in vitro Induction of endogenous miR-9 expression, upon ligand-dependent stimulation of PDGFRβ signaling, promoted significant vascular sprouting of TNBC cells, in part, by direct repression of STARD13. Conversely, ectopic expression of miR-200 inhibited this sprouting by indirectly reducing the protein levels of PDGFRβ through the direct suppression of ZEB1. Notably, in vivo miR-9 inhibition or miR-200c restoration, through either the generation of MDA-MB-231-stable clones or peritumoral delivery in MDA-MB-231 xenografted mice, strongly decreased the number of vascular lacunae. Finally, IHC and immunofluorescence analyses in TNBC specimens indicated that PDGFRβ expression marked tumor cells engaged in vascular lacunae. In conclusion, our results demonstrate that miR-9 and miR-200 play opposite roles in the regulation of the vasculogenic ability of TNBC, acting as facilitator and suppressor of PDGFRβ, respectively. Moreover, our data support the possibility to therapeutically exploit miR-9 and miR-200 to inhibit the process of vascular lacunae formation in TNBC. Cancer Res; 76(18); 5562-72. ©2016 AACR.

Overview of diagnostic/targeted treatment combinations in personalized medicine for breast cancer patients

Breast cancer includes a body of molecularly distinct subgroups, characterized by different presentation, prognosis, and sensitivity to treatments. Significant advances in our understanding of the complex architecture of this pathology have been achieved in the last few decades, thanks to new biotechnologies that have recently come into the research field and the clinical practice, giving oncologists new instruments that are based on biomarkers and allowing them to set up a personalized approach for each individual patient. Here we review the main treatments available or in preclinical development, the biomolecular diagnostic and prognostic approaches that changed our perspective about breast cancer, giving an overview of targeted therapies that represent the current standard of care for these patients. Finally, we report some examples of how new technologies in clinical practice can set in motion the development of new drugs.

Ran Binding Protein 9 (RanBP9) is a novel mediator of cellular DNA damage response in lung cancer cells

Ran Binding Protein 9 (RanBP9, also known as RanBPM) is an evolutionary conserved scaffold protein present both in the nucleus and the cytoplasm of cells whose biological functions remain elusive. We show that active ATM phosphorylates RanBP9 on at least two different residues (S181 and S603). In response to IR, RanBP9 rapidly accumulates into the nucleus of lung cancer cells, but this nuclear accumulation is prevented by ATM inhibition. RanBP9 stable silencing in three different lung cancer cell lines significantly affects the DNA Damage Response (DDR), resulting in delayed activation of key components of the cellular response to IR such as ATM itself, Chk2, γH2AX, and p53. Accordingly, abrogation of RanBP9 expression reduces homologous recombination-dependent DNA repair efficiency, causing an abnormal activation of IR-induced senescence and apoptosis. In summary, here we report that RanBP9 is a novel mediator of the cellular DDR, whose accumulation into the nucleus upon IR is dependent on ATM kinase activity. RanBP9 absence hampers the molecular mechanisms leading to efficient repair of damaged DNA, resulting in enhanced sensitivity to genotoxic stress. These findings suggest that targeting RanBP9 might enhance lung cancer cell sensitivity to genotoxic anti-neoplastic treatment.

Role of BAX for outcome prediction in gastrointestinal malignancies

Our group and numerous others have shown in both preclinical and clinical studies that the proapoptotic mediator BAX may be deregulated through gene mutation or loss of protein expression, affecting resistance to chemotherapy and radiotherapy in several cancer types. However, BAX is also involved in cancer development and may related to prognosis, independently of treatment outcome. The clinical impact of BAX status in gastrointestinal malignancies remains controversial, although it is generally hypothesized that high expression may be a positive prognostic factor and predict increased efficacy of chemotherapy (with particular regard to platinum derivatives). The present review aims to provide updated information on BAX as potential prognostic and/or predictive biomarker in gastroesophageal and colorectal cancers, as well as in other less studied gastrointestinal malignancies.

Phase I study of tomuzotuximab, a glycoengineered therapeutic antibody against the epidermal growth factor receptor, in patients with advanced carcinomas

Background Changes in glycosylation of the constant domain (Fc) of monoclonal antibodies (mAbs) enhance antibody-dependent cell-mediated cytotoxicity independently of downstream effects following receptor blockade by the antibody, thus extending their indication. We investigated the safety, pharmacokinetics, pharmacodynamics and antitumour activity of tomuzotuximab, an IgG1 glycoengineered mAb against the epidermal growth factor receptor with enhanced tumour cytotoxicity in a phase I dose-escalation study (NTC01222637). Patients and methods Forty-one patients with advanced solid tumours refractory to standard therapies received tomuzotuximab weekly (12–1370 mg) or two-weekly (990 mg) on a three-plus-three dose escalation design. Results A maximum tolerated dose was not reached. The most frequent treatment-related adverse events were infusion-related reactions in 31 (76%) patients (grade 3, 12%), mainly confined to the first dose, and skin toxicities (grade 1 or 2) in 30 (73%) patients. Hypomagnesaemia was observed in 9 out of 23 evaluable patients (39%). Similar to cetuximab, tomuzotuximab concentrations increased proportionally to dose from doses≥480 mg with a median terminal half life (t½) of 82 hours, range 55–113 hours. Antitumour activity included one complete response ongoing since more than 4.5 years in a patient with non-small-cell lung cancer and one partial response lasting 353 days in a patient with colorectal cancer. Twelve patients achieved stable disease (median, 166 days, range, 71–414 days) and two patients had prolonged control (>1 year) of their non-measurable disease. Conclusion Tomuzotuximab was safe and showed promising antitumour activity in heavily pretreated patients with advanced metastatic disease. A phase IIb trial of chemotherapy and weekly tomuzotuximab or cetuximab followed with maintenance therapy with the corresponding mAb in patients with recurrent or metastatic head and neck squamous cell carcinoma is ongoing.

Genetic ablation of interacting with Spt6 (Iws1) causes early embryonic lethality

IWS1 is an RNA-polymerase II (RNAPII)-associated transcription elongation factor whose biological functions are poorly characterized. To shed some light on the function of this protein at the organismal level, we performed a systematic tissue analysis of its expression and generated Iws1-deficient mice. A thorough immunohistochemical characterization shows that IWS1 protein is present in the nucleus of all cells in most of the examined tissues, with few notable exceptions. We also report that ablation of Iws1 consistently causes lethality at the pre-implantation stage with high expression of the gene in fertilized oocytes. In summary, we are providing evidence that Iws1 is expressed in all adult organs and it is an essential gene for mouse embryonic development.

RANBP9 affects cancer cells response to genotoxic stress and its overexpression is associated with worse response to platinum in NSCLC patients

Although limited by severe side effects and development of resistance, platinum-based therapies still represent the most common first-line treatment for non-small cell lung cancer (NSCLC). However, a crucial need in the clinical management of NSCLC is represented by the identification of cases sensitive to DNA damage response (DDR)-targeting drugs, such as cisplatin or PARP inhibitors. Here, we provide a molecular rationale for the stratification of NSCLC patients potentially benefitting from platinum compounds based on the expression levels of RANBP9, a recently identified player of the cellular DDR. RANBP9 was found overexpressed by immunohistochemistry (IHC) in NSCLC compared to normal adjacent tissues (NATs) (n = 147). Moreover, a retrospective analysis of 132 platinum-treated patients from the multi-centric TAILOR trial showed that RANBP9 overexpression levels are associated with clinical response to platinum compounds [Progression Free Survival Hazard Ratio (RANBP9 high vs low) 1.73, 95% CI 1.15–2.59, p = 0.0084; Overall Survival HR (RANBP9 high vs low) 1.99, 95% CI 1.27–3.11, p = 0.003]. Accordingly, RANBP9 KO cells showed higher sensitivity to cisplatin in comparison with WT controls both in vitro and in vivo models. NSCLC RANBP9 KO cells were also more sensitive than control cells to the PARP inhibitor olaparib alone and in combination with cisplatin, due to defective ATM-dependent and hyper-activated PARP-dependent DDR. The current investigation paves the way to prospective studies to assess the clinical value of RANBP9 protein levels as prognostic and predictive biomarker of response to DDR-targeting drugs, leading to the development of new tools for the management of NSCLC patients.

Scorpins in the DNA Damage Response

The DNA Damage Response (DDR) is a complex signaling network that comes into play when cells experience genotoxic stress. Upon DNA damage, cellular signaling pathways are rewired to slow down cell cycle progression and allow recovery. However, when the damage is beyond repair, cells activate complex and still not fully understood mechanisms, leading to a complete proliferative arrest or cell death. Several conventional and novel anti-neoplastic treatments rely on causing DNA damage or on the inhibition of the DDR in cancer cells. However, the identification of molecular determinants directing cancer cells toward recovery or death upon DNA damage is still far from complete, and it is object of intense investigation. SPRY-containing RAN binding Proteins (Scorpins) RANBP9 and RANBP10 are evolutionarily conserved and ubiquitously expressed proteins whose biological functions are still debated. RANBP9 has been previously implicated in cell proliferation, survival, apoptosis and migration. Recent studies also showed that RANBP9 is involved in the Ataxia Telangiectasia Mutated (ATM) signaling upon DNA damage. Accordingly, cells lacking RANBP9 show increased sensitivity to genotoxic treatment. Although there is no published evidence, extensive protein similarities suggest that RANBP10 might have partially overlapping functions with RANBP9. Like RANBP9, RANBP10 bears sites putative target of PIK-kinases and high throughput studies found RANBP10 to be phosphorylated following genotoxic stress. Therefore, this second Scorpin might be another overlooked player of the DDR alone or in combination with RANBP9. This review focuses on the relatively unknown role played by RANBP9 and RANBP10 in responding to genotoxic stress.

MP83-15 A NOVEL THERAPY FOR CASTRATION-RESISTANT PROSTATE CANCER THROUGH INHIBITION OF ONCOGENIC MICRORNAS

INTRODUCTION AND OBJECTIVES: Management of castration-resistant prostate cancer (CPRC) remains challenging due to the inevitable emergence of resistance to treatments including radiotherapy (RT) and chemotherapy (CT). We previously reported that zoledronic acid (ZOL) clinically potentiates the antitumor effects of RT in patients with renal cell carcinoma (Kijima et al, BJU Int 2009) and that this radiosensitization could occur through the osteoclast-independent inhibition of signal transducer and activator of transcription 1 (STAT1) (Kijima et al, PLoS One 2013). As the association between STAT1 overexpression and treatment resistance has been reported in several cancers, we investigated whether STAT1 is associated with resistance to RT and CT in CRPC cells and whether ZOL could overcome this resistance by downregulating STAT1. METHODS: Baseline expression of STAT1 was compared between androgen-dependent LNCaP cells and androgen-independent LNCaP (LNCaP-CR), PC3, and DU145 cells. The effect of ZOL on STAT1 expression was evaluated by Western blot and real-time PCR. The sensitizing effects of ZOL on RT and CT (docetaxel) were examined by clonogenic assay and MTS assay with combination index analysis. To confirm the importance of STAT1 on radioand chemosensitization by ZOL, both siRNA knockdown and forced expression by cDNA transfection were performed. RESULTS: STAT1 levels were higher in androgen-independent cell lines (PC3, DU145) than in LNCaP cells (Figure A). STAT1 was gradually upregulated in LNCaP as these cells acquired androgen independency through continuous androgen ablation (Figure B). ZOL decreased STAT1 at the protein level (Figure C) through proteasomemediated degradation and sensitized PC3 and DU145 to both RT and CT. Functional siRNA knockdown of STAT1 resulted in the sensitization of DU145 to RT and CT. Forced expression of STAT1 in LNCaP cells rendered them resistant to those therapies. CONCLUSIONS: ZOL sensitizes CRPC cells to RT and CT by downregulating STAT1.

Disease-free interval in metastatic breast cancer patients undergoing complete remission: implications of cross-sectional study design and biologic considerations about disease history

We thank Dr. Altundag for the interesting comment to our paper about complete response in metastatic breast cancer [1]. In his ‘‘Letter to the Editor’’, he points out that long disease-free interval may have favorably influenced the incidence of complete responses observed, due to its independent positive prognostic value [2]. At first, we would like to underline that the design of our work (cross-sectional cohort retrospective study) does not allow to make causality or association inferences. In fact, as we clarified in the discussion of results, it does not suit the purpose of identifying potential predictive factors of complete response. It only presents a descriptive analysis of a monocentric case series and its results are exquisitely hypothesis generating [3]. Moreover we wish to highlight that the described population was unselected, as we collected all cases of complete response among metastatic breast cancer patients treated at our Institution in a pre-defined range of time. Actually, baseline characteristics of study population reflect what can be found in common clinical practice (e.g. 31.6% of cases metastatic at diagnosis, 82.4% of cases with hormone receptor positive tumor, 35.1% of cases overexpressing HER2). Nonetheless, we confirm what we stated in the paper: study design itself presents an intrinsic selection bias, in that only alive patients at the time of enrollment have been analyzed and this may have entailed exclusion of cases with poor survival. Going beyond these methodological issues, we cannot completely endorse the point that a longer disease-free interval may be associated with a higher frequency of complete responses. A consistent amount of literature suggests that indolent disease is related to a lower response rate to medical treatments. Most of these data have been obtained in neoadjuvant setting, where pathologic indices of indolent biology (e.g. luminal-like phenotype, low proliferation rate, well differentiated histology) are associated with a lower incidence of pathologic complete response [4]. The assumption that indolent disease is less responsive to therapies can be plausibly translated in metastatic setting as well. Consequently, we may hypothesize that aggressive, highly proliferative disease is more likely to show a complete remission following systemic treatment. In conclusion, we restate that further studies are needed to ascertain patients’ and tumor characteristics potentially predictive of complete response to medical therapy.