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Supplementary Figure legends (S1-S4)

MiR-9 regulates PDGFRbeta expression in TNBC cell lines

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.

The file contains all the supplementary tables (S1-S4): - Supplementary Table S1. Clinicopathologic features of 78 breast cancer patients (set 1) and 85 TNBC (set 2). - Supplementary Table S2. Relation between miR-9 expression and clinicopathologic characteristics in human breast cancer. - Supplementary Table S3. Top 40 genes predicted as miR-9 target and statistically significant down-modulated in basal versus luminal breast cancer. - Supplementary Table S4. Relation between clinicopathologic characteristics and miR-9 and miR-200c in TNBC human tissues.

Importance. Neoadjuvant therapy with dual HER2 blockade improved pathological complete response (pCR) rate. Nevertheless, it would be desirable to identify patients exquisitely responsive to single agent trastuzumab to minimize or avoid overtreatment. Objective. To evaluate the predictive and prognostic value of basal primary tumor miRNA expression profile within the trastuzumab arm of the NeoALTTO study.Design, Setting and Participants. RNA samples from baseline biopsies were randomized into training (n =45) and testing (n =47) sets. After normalization, miRNAs associated with Event-free survival (EFS) and pCR were identified by univariate analysis. Multivariate models were implemented to generate specific signatures which were first confirmed, and then analyzed according to other clinical and pathological variables.Main outcomes and measures. Association between miRNA expression and pCR and/or EFS.Results. We identified a prognostic signature including hsa-miR-153-3p (HR 1.831, 95%CI: 1.34-2.50) and hsa-miR-219a-5p (HR 0.629, 95%CI: 0.50 - 0.78). For two additional miRNAs (miR-215-5p and miR-30c-2-3p), we found a statistically significant interaction term with pCR (p.interaction: 0.017 and 0.038, respectively). Besides, a two-miRNA signature was predictive of pCR (hsa-miR-31-3p, OR 0.70, 95%CI: 0.53 - 0.92, and hsa-miR-382-3p, OR: 1.39, 95%CI: 1.01 -1.91). Notably, the performance of this predictive miRNA signature resembled that of the genomic classifiers PAM50 and TRAR, and did not improve when the extended models were fitted.Conclusions and relevance. Analysis of primary tumor tissue miRNAs holds the potential of a parsimonious tool to identify patients with differential clinical outcomes after trastuzumab based neoadjuvant therapy. Trial registration. ClinicalTrials.gov Identifier: NCT00553358. Table 1.MiRNAs associated with EFS. Results of the univariate Cox regression model- training set.miRNAsHR95% CIhsa-miR-12000.671(0.512; 0.879)ahsa-miR-1238-3p0.669(0.543; 0.826)ahsa-miR-12650.839(0.705; 0.997)ahsa-miR-129-5p0.785(0.628; 0.980)ahsa-miR-153-3p1.412(1.026; 1.943)ahsa-miR-15390.826(0.697; 0.979)ahsa-miR-1908-5p0.796(0.648; 0.978)ahsa-miR-205-5p0.737(0.553; 0.982)hsa-miR-219a-5p0.787(0.627; 0.989)ahsa-miR-25-5p0.566(0.391; 0.819)ahsa-miR-3000.581(0.399; 0.845)ahsa-miR-382-3p0.791(0.636; 0.985)ahsa-miR-4920.774(0.635; 0.944)ahsa-miR-519a-3p0.834(0.696; 0.998)ahsa-miR-551b-5p0.666(0.452; 0.981)ahsa-miR-5830.727(0.549; 0.963)ahsa-miR-6001.373(1.03; 1.829)ahsa-miR-6260.819(0.673; 0.998)ahsa-miR-651-5p0.611(0.382; 0.977)hsa-miR-7610.845(0.721; 0.991)ahsa-miR-891b0.658(0.479; 0.904)ahsa-miR-92a-2-5p0.787(0.637; 0.971)ahsa-miR-937-3p0.748(0.578; 0.967)aHR, Hazard Ratio; CI, Confidence Intervala microRNAs retained its statistically significance (at 10%) also when normalized for the overall mean. Table 2.MiRNAs associated with pCR. Results of the univariate logistic model - training set.miRNAsOR95% CIhsa-miR-132-3p0.410(0.169; 0.991)ahsa-miR-23b-5p0.520(0.316; 0.856)ahsa-miR-31-3p0.566(0.351; 0.912)ahsa-miR-31-5p0.508(0.291; 0.887)ahsa-miR-330-3p0.501(0.251; 0.999)hsa-miR-34b-3p0.673(0.474; 0.956)ahsa-miR-382-3p1.392(1.006; 1.925)ahsa-miR-548j-5p1.702(1.090; 2.658)aOR, Odds Ratio; CI, Confidence Interval. a microRNAs retained its statistically significance (at 10%) also when normalized for the overall mean. 2 Citation Format: Marilena Valeria Iorio, Sara Pizzamiglio, Giulia Cosentino, Chiara M Ciniselli, Loris De Cecco, Alessandra Cataldo, Ilaria Plantamura, Tiziana Triulzi, Sarra El-abed, Yingbo Wang, Mohammed Bajji, Paolo Nuciforo, Jens Huober, Susan Ellard, David Rimm, Andrea Gombos, Mariagrazia Daidone, Paolo Verderio, Elda Tagliabue, Serena Di Cosimo. The future of HER2-positive breast cancer patients might be written in miRNAs: An exploratory analysis from the NeoALTTO study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-26.

Breast cancer that highly expresses human epidermal growth factor receptor 2 (HER2+) represents one of the major breast cancer subtypes, and was associated with a poor prognosis until the introduction of HER2-targeted therapies such as trastuzumab. Unfortunately, up to 30% of patients with HER2+ localized breast cancer continue to relapse, despite treatment. MicroRNAs (miRNAs) are small (approximately 20 nucleotides long) non-coding regulatory oligonucleotides. They function as post-transcriptional regulators of gene expression, binding complementarily to a target mRNA and leading to the arrest of translation or mRNA degradation. In the last two decades, translational research has focused on these small molecules because of their highly differentiated expression patterns in blood and tumor tissue, as well as their potential biological function. In cancer research, they have become pivotal for the thorough understanding of oncogenic biological processes. They might also provide an efficient approach to early monitoring of tumor progression or response to therapy. Indeed, changes in their expression patterns can represent a flag for deeper biological changes. In this review, we sum up the recent literature regarding miRNAs in HER2+ breast cancer, taking into account their potential as powerful prognostic and predictive biomarkers, as well as therapeutic tools.

The implementation of trastuzumab has revolutionized the clinical management of HER2 positive breast cancers. Unfortunately, 50% of patients are resistant to the treatment. Researchers have already designed alternative anti-HER2 agents, such as pertuzumab and lapatinib. Still, predicting which patients will benefit from the therapy would prevent overtreatment and avoid unnecessary risks of side effects. MiRNAs are small non-coding RNAs involved in post-transcriptional gene regulation, and participate in almost all biological processes, including cancer. Since aberrant miRNA levels can be detected both at tissue level and in the circulation, they are good candidates as predictive and prognostic biomarkers. This study had access to tumor tissue samples from the phase III NeoALTTO trial, aimed at evaluating the efficacy of a HER2 dual blockade with trastuzumab and Lapatinib vs single blocking, in concomitance with chemotherapy, in a pre-operative setting. The primary end-point of the study was pathologic complete response (pCR); the secondary end-point event-free survival (EFS). Focusing on the trastuzumab arm, we identified both a predictive signature (hsa-miR-31-3p, OR 0.70, 95%CI: 0.53-0.92 and hsa-miR-382-3p, OR 1.39, 95%CI: 1.01-1.91) with an AUC value of 0.73 (95%CI: 0.60-0.87), and a prognostic signature (miR-153-3p, HR 1.83, 95%CI: 1.34-2.50 and miR-219a-5p, HR 0.629, 95%CI: 0.51-0.79) leading to a C-statistics of 0.730 (95%CI: 0.63-0.83). Moreover, we identified 2 miRNAs (miR-215-5p and miR-30c-2-3p) associated to EFS with a statistically significant interaction term with pCR (p.interaction: 0.017 and 0.038, respectively). Aiming at deepening the understanding of resistance mechanism, we modulated the expression of miR-31-3p and miR-382-3p in vitro, upon trastuzumab treatment in HER2 positive breast cancer cell lines. Given that miR-31-3p negatively correlates with pCR, we transfected it in HER2-addicted SKBr3 cells to appreciate a possible gain of resistance to trastuzumab; conversely, miR-382-3p positively correlates with pCR and was transfected in HER2 non-addicted HCC1954 cells. Western blot analysis of HER2 signaling pathway highlighted that overexpression of miR-31-3p was able to counteract the reduction of phosphorylated HER2 levels induced by trastuzumab treatment in SKBR3 cells compared to control. Interestingly, miR-31-3p upmodulation increased the proliferation of both treated and non-treated SKBr3 cell in a 3D setting. Conversely, miR-382-3p overexpression in HCC1954 cells only slightly increased responsiveness to trastuzumab in the 3D setting, compared to control. Trastuzumab efficacy also relies on the immune system reaction, thus, in vivo experiments will likely provide further insights into the mechanism of action of these miRNAs. Citation Format: Giulia Cosentino, Sara Pizzamiglio, Chiara M. Ciniselli, Loris De Cecco, Alessandra Cataldo, Ilaria Plantamura, Tiziana Triulzi, Sarra El-abed, Yingbo Wang, Mohammed Bajji, Paolo Nuciforo, Jens Huober, Susan L. Ellard, David L. Rimm, Andrea Gombos, Mariagrazia Daidone, Paolo Verderio, Elda Tagliabue, Serena Di Cosimo, Marilena V. Iorio. miRNA and response to trastuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1229.

Tumor growth and invasion occurs through a dynamic interaction between cancer and stromal cells, which support an aggressive niche. MicroRNAs are thought to act as tumor messengers to &ldquo, corrupt&rdquo, stromal cells. We previously demonstrated that miR-9, a known metastamiR, is released by triple negative breast cancer (TNBC) cells to enhance the transition of normal fibroblasts (NFs) into cancer-associated fibroblast (CAF)-like cells. EGF containing fibulin extracellular matrix protein 1 (EFEMP1), which encodes for the ECM glycoprotein fibulin-3, emerged as a miR-9 putative target upon miRNA&rsquo, s exogenous upmodulation in NFs. Here we explored the impact of EFEMP1 downmodulation on fibroblast&rsquo, s acquisition of CAF-like features, and how this phenotype influences neoplastic cells to gain chemoresistance. Indeed, upon miR-9 overexpression in NFs, EFEMP1 resulted downmodulated, both at RNA and protein levels. The luciferase reporter assay showed that miR-9 directly targets EFEMP1 and its silencing recapitulates miR-9-induced pro-tumoral phenotype in fibroblasts. In particular, EFEMP1 siRNA-transfected (si-EFEMP1) fibroblasts have an increased ability to migrate and invade. Moreover, TNBC cells conditioned with the supernatant of NFs transfected with miR-9 or si-EFEMP1 became more resistant to cisplatin. Overall, our results demonstrate that miR-9/EFEMP1 axis is crucial for the conversion of NFs to CAF-like cells under TNBC signaling.

Introduction: Chemotherapy is still the standard of care for triple-negative breast cancers (TNBCs). Here, we investigated miR-302b as a therapeutic tool to enhance cisplatin sensitivity in vivo and unraveled the molecular mechanism. Materials and Methods: TNBC-xenografted mice were treated with miR-302b or control, alone or with cisplatin. Genome-wide transcriptome analysis and independent-validation of Integrin Subunit Alpha 6 (ITGA6) expression was assessed on mice tumor samples. Silencing of ITGA6 was performed to evaluate cisplatin response in vitro. Further, potential transcription factors of ITGA6 (E2F transcription facor 1 (E2F1), E2F transcription factor 2 (E2F2), and Yin Yang 1 (YY1)) were explored to define the miRNA molecular mechanism. The miR-302b expression was also assessed in TNBC patients treated with chemotherapy. Results: The miR&ndash, 302b-cisplatin combination significantly impaired tumor growth versus the control through indirect ITGA6 downregulation. Indeed, ITGA6 was downmodulated in mice treated with miR-302b&ndash, cisplatin, and ITGA6 silencing increased drug sensitivity in TNBC cells. In silico analyses and preclinical assays pointed out the regulatory role of the E2F family and YY1 on ITGA6 expression under miR-302b&ndash, cisplatin treatment. Finally, miR-302b enrichment correlated with better overall survival in 118 TNBC patients. Conclusion: MiR-302b can be exploited as a new therapeutic tool to improve the response to chemotherapy, modulating the E2F family, YY1, and ITGA6 expression. Moreover, miR-302b could be defined as a new prognostic factor in TNBC patients.

Trastuzumab is the standard treatment for HER2+ breast cancer (BC) patients, and even though it significantly improved their clinical outcome, 50% of them do not benefit from this drug and disease recurs, underlining the need of reliable predictive biomarkers and new therapeutic strategies. Strikingly, despite all the molecular analyses performed to identify the escape mechanisms behind this resistance, it still represents a question point. MiRNAs have been correlated with occurrence and progression of human cancer, and their potential as clinical tools has emerged in the last years. We previously reported that oncosuppressive miR-205 targets HER3, thus increasing the responsiveness to TKIs lapatinib and gefitinib in preclinical models. Here we demonstrate that HER3 inhibition by miR-205 ectopic expression or siRNA-mediated silencing improves the responsiveness to Trastuzumab in vitro in HER2+ BC cell lines, and that this effect is exerted through impairment of AKT-mediated pathway. Moreover, evaluating a series of 52 HER2+ BC patients treated with adjuvant Trastuzumab, we observed that higher miR-205 expression is significantly associated with better outcome (disease-free survival). In summary, our data indicate that miR-205 could predict Trastuzumab efficacy and that its modulation might be useful as adjuvant treatment to improve the response to the drug.

Simple Summary The cross-talk between neoplastic cells and microenvironment is known to play a crucial role in tumor development as well as in the phenomenon of resistance to anticancer therapies. MicroRNAs, involved in the pathogenesis of human tumors, are among the molecules exploited in this aberrant cross-talk. Indeed, microRNAs play a crucial function both in the induction of pro-tumoral traits in stromal cells and in the stroma-mediated fueling of tumor aggressiveness. Here, we reviewed the most recent literature concerning the role of miRNAs in shaping the tumor microenvironment, and the consequences on responsiveness to therapies. Abstract The clinical management of breast cancer reaches new frontiers every day. However, the number of drug resistant cases is still high, and, currently, this constitutes one of the major challenges that cancer research has to face. For instance, 50% of women affected with HER2 positive breast cancer presents or acquires resistance to trastuzumab. Moreover, for patients affected with triple negative breast cancer, standard chemotherapy is still the fist-line therapy, and often patients become resistant to treatments. Tumor microenvironment plays a crucial role in this context. Indeed, cancer-associated stromal cells deliver oncogenic cues to the tumor and vice versa to escape exogenous insults. It is well known that microRNAs are among the molecules exploited in this aberrant crosstalk. Indeed, microRNAs play a crucial function both in the induction of pro-tumoral traits in stromal cells and in the stroma-mediated fueling of tumor aggressiveness. Here, we summarize the most recent literature regarding the involvement of miRNAs in the crosstalk between tumor and stromal cells and their capability to modulate tumor microenvironment characteristics. All up-to-date findings suggest that microRNAs in the TME could serve both to reverse malignant phenotype of stromal cells, modulating response to therapy, and as predictive/prognostic biomarkers.

CDCP1, a transmembrane noncatalytic receptor, the expression of which has been associated with a poor prognosis in certain epithelial cancers, was found to be expressed in highly aggressive triple-negative breast cancer (TNBC) cell models, in which it promoted aggressive activities-ie, migration, invasion, anchorage-independent tumor growth, and the formation of vascular-like structures in vitro. By immunohistochemical (IHC) analysis of 100 human TNBC specimens, CDCP1 was overexpressed in 57% of samples, 38% of which exhibited a gain in CDCP1 copy number by fluorescence in situ hybridization (FISH). CDCP1 positivity was significantly associated between FISH and IHC. CDCP1 expression and gains in CDCP1 copy number synergized with nodal (N) status in determining disease-free and distant disease-free survival. The hazard ratios (HRs) of the synergies between CDCP1 positivity by IHC and FISH and lymph node positivity in predicting relapse did not differ significantly, indicating that CDCP1 overexpression in human primary TNBCs, regardless of being driven by gains in CDCP1, is for a critical factor in the progression of N-positive TNBCs. Thus, CDCP1 is a novel marker of the most aggressive N-positive TNBCs and a potential therapeutic target.

MicroRNAs are a class of small non-coding regulatory RNAs playing key roles in cancer. Breast cancer is the most common female malignancy worldwide and is categorized into four molecular subtypes: luminal A and B, HER2+ and triple-negative breast cancer (TNBC). Despite the development of multiple targeted therapies for luminal and HER2+ breast tumors, TNBC lacks specific therapeutic approaches, thus they are treated mainly with radio- and chemotherapy. The effectiveness of these therapeutic regimens is based on their ability to induce DNA damage, which is differentially resolved and repaired by normal vs. cancer cells. Recently, drugs directly targeting DNA repair mechanisms, such as PARP inhibitors, have emerged as attractive candidates for the future molecular targeted-therapy in breast cancer. These compounds prevent cancer cells to appropriate repair DNA double strand breaks and induce a phenomenon called synthetic lethality, that results from the concurrent inhibition of PARP and the absence of functional BRCA genes which prompt cell death. MicroRNAs are relevant players in most of the biological processes including DNA damage repair mechanisms. Consistently, the downregulation of DNA repair genes by miRNAs have been probe to improve the therapeutic effect of genotoxic drugs. In this review, we discuss how microRNAs can sensitize cancer cells to DNA-damaging drugs, through the regulation of DNA repair genes, and examine the most recent findings on their possible use as a therapeutic tools of treatment response in breast cancer.

Oxidative stress is a pathological condition determined by a disturbance in reactive oxygen species (ROS) homeostasis. Depending on the entity of the perturbation, normal cells can either restore equilibrium or activate pathways of cell death. On the contrary, cancer cells exploit this phenomenon to sustain a proliferative and aggressive phenotype. In fact, ROS overproduction or their reduced disposal influence all hallmarks of cancer, from genome instability to cell metabolism, angiogenesis, invasion and metastasis. A persistent state of oxidative stress can even initiate tumorigenesis. MicroRNAs (miRNAs) are small non coding RNAs with regulatory functions, which expression has been extensively proven to be dysregulated in cancer. Intuitively, miRNA transcription and biogenesis are affected by the oxidative status of the cell and, in some instances, they participate in defining it. Indeed, it is widely reported the role of miRNAs in regulating numerous factors involved in the ROS signaling pathways. Given that miRNA function and modulation relies on cell type or tumor, in order to delineate a clearer and more exhaustive picture, in this review we present a comprehensive overview of the literature concerning how miRNAs and ROS signaling interplay affects breast cancer progression.

miR-9 has been described as an oncogenic microRNA associated to a metastatic phenotype and able to induce EMT (epithelial-to-mesenchymal transition) through direct targeting of E-cadherin. However, data available concerning the expression and the role of this microRNA in different subgroups of breast cancer are still not exhaustive. Evaluating miR-9 expression by Real-Time PCR in a series of 92 breast cancer specimens (35 luminal, 36 HER2, 21 triple negative), we found that this microRNA is increasingly higher in HER2 and Triple Negative versus ER positive patients (fold change 3 and 8 respectively). Moreover, preliminary analysis of miR-9 expression in correlation with bio-pathological features and clinical data also indicates a trend in association with disease progression. Triple Negative Breast Cancers represent a very aggressive breast cancer subgroup, still lacking specific markers for an effective targeted therapy; we investigated whether miR-9 might play a role in the biology of this tumor subtype. Preliminary data indicate that miR-9 is activated downstream PDGFRbeta, which represents a crucial player in the aggressive phenotype of Triple Negative Breast Cancer. In summary, here we show that miR-9 is significantly upregulated in triple negative breast cancer in comparison with other breast cancer subgroups and is activated downstream PDGFRbeta. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-18.

Introduction: Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer cases, with the worst outcome of all subtypes. For TNBC, still lacking targeted therapies, the only therapeutic option is currently chemotherapy, and despite a good initial response, patients often develop drug resistance. MiRNAs can modulate chemoresistance by affecting DNA repair, cell cycle progression, apoptosis and also tumor microenvironment. Macrophages constitute a major component of the immune microenvironment of cancer and pro-tumor M2 macrophages have been associated with resistance to chemotherapeutic treatments. Our previous data showed that miR-302b over-expression enhances sensitivity to cisplatin in breast cancer cell lines by targeting directly E2F1 and indirectly ATM. Here, we investigated the potential of miR-302b as a therapeutic tool to enhance cisplatin response in a TNBC mouse model and which pathways are involved in this mechanism both in tumor cells and microenvironment. Moreover, miR-302b prognostic value was assessed in a cohort of TNBC patients with available clinical outcome . Finally, we evaluated if miR-302b enhances the sensitivity to doxorubicin, another chemotherapeutic agent used as first-line therapy in TNBC patients. Material and method: MDA-MB-231 TNBC cells were injected into the mammary fat pad of female SCID mice and then treated with lipid nanoparticles containing miR-302b or cel-miR-67 control, alone or in combination with cisplatin. Gene expression profile on collected tumors was performed by microarray and tumor sections were stained with anti-arginase 1 (M2 marker) to evaluate the number of M2 macrophages. MiR-302b expression was assessed in 39 TNBC treated with chemotherapy in adjuvant setting, and associated with prognosis. Finally, MDA-MB-231 cells were transfected with miR-302b precursor or control treated with doxorubicin for 24h and then assessed for cell viability. Results: Our results show that miR-302b combination with cisplatin significantly impaired tumor growth in comparison with cel-67 control and cisplatin (p= 0.03), and reduced the number of M2 macrophages in the tumor microenvironment (p=0.005). Moreover, gene expression profile of collected tumors confirm immune system modulation. Notably, miR-302b expression was associated with disease-free survival and overall survival in TNBC patients treated with adjuvant chemotherapy. Furthermore, we found that miR-302b also enhances sensitivity to doxorubicin in vitro, affecting cell viability and cell cycle transition through E2F1 regulation. Conclusion: Our data demonstrate that miR-302b can be exploited as a new therapeutic tool to improve the response to chemotherapy, modulating tumor microenvironment. Moreover, this miRNA has prognostic significance in TNBC patients, and might also represent an useful predictive biomarker for response to chemotherapy. Citation Format: Alessandra Cataldo, Ilaria Plantamura, Elvira D'Ippolito, Sandra Romero-Cordoba, Sara Baroni, Valeria Cancilia, Claudio Tripodo, Dario Palmieri, Marilena V. Iorio. miR-302b as adjuvant therapeutic tool to improve chemotherapy efficacy in human triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5437. doi:10.1158/1538-7445.AM2017-5437

Triple negative breast cancer (TNBC) is a very aggressive subgroup of breast carcinoma, still lacking specific markers for an effective targeted therapy and with a poorer prognosis compared to other breast cancer subtypes. In this study we investigated the possibility that TNBC cells contribute to the establishment of tumor vascular network by the process known as vasculogenic mimicry, through endothelial cell differentiation. Vascular-like functional properties of breast cancer cell lines were investigated in vitro by tube formation assay and in vivo by confocal microscopy, immunofluorescence or immunohistochemistry on frozen tumor sections. TNBCs express endothelial markers and acquire the ability to form vascular-like channels in vitro and in vivo, both in xenograft models and in human specimens, generating blood lacunae surrounded by tumor cells. Notably this feature is significantly associated with reduced disease free survival. The impairment of the main pathways involved in vessel formation, by treatment with inhibitors (i.e. Sunitinib and Bevacizumab) or by siRNA-mediating silencing, allowed the identification of PDGFRβ and FGFR2 as relevant players in this phenomenon. Inhibition of these tyrosine kinase receptors negatively affects vascular lacunae formation and significantly inhibits TNBC growth in vivo. In summary, we demonstrated that TNBCs have the ability to form vascular-like channels in vitro and to generate blood lacunae lined by tumor cells in vivo. Moreover, this feature is associated with poor outcome, probably contributing to the aggressiveness of this breast cancer subgroup. Finally, PDGFRβ and FGFR2-mediated pathways, identified as relevant in mediating this characteristic, potentially represent valid targets for a specific therapy of this breast cancer subgroup.

Background. Triple Negative Breast Cancers (TNBCs) still represent a question mark in breast cancer biology and a primary issue in clinics. A promising approach for an efficient targeted treatment of TNBCs seems to be represented by antiangiogenic therapies. Methods. Effects of different compounds on proliferation of TNBC was evaluated in vivo and in vitro respectively by xenograft tumor volume analysis and SRB assay. Vasculogenic Mimicry (VM) properties were evaluated in vitro and in vivo respectively by tube formation assay and Transmission Electron Microscopy (TEM). Results. Treatment of triple negative (TN) MDA-MB-231 xenografts with Sunitinib induced tumor regression, versus only a slight growth inhibition in tumors derived from MCF7 luminal cell line, whereas Bevacizumab determined only a modest decrease in tumor growth in both models. Accordingly, the efficacy of Sunitinib in blocking in vitro growth of breast cancer cell lines was higher in MDA-MB-231 cells in comparison with MCF7 cells (IC50 at 72h in MDA-MB-231 was 5 uM vs 25 uM in MCF7, as evaluated by SRB), and sensitivity to Bevacizumab was comparable between the two different cell lines. Investigating the undifferentiated nature of TNBCs, we observed that these tumors present an endothelial like phenotype and behavior, as supported respectively by the expression of endothelial markers, and the formation of vascular-like channels in vitro. In fact, all six TN cell lines evaluated (MDA-MB-231, MDA-MB-157, MDA-MB-468, BT-549, BT-20 and HCC1937) were able to form vascular channels when seeded on the murine tumor-derived basement membrane (Matrigel), whereas luminal (MCF7, T47D and ZR-75-1) and HER2- positive (MDA-MB-361, BT474 and SKBr3) breast carcinoma cell lines did not exhibit vascular structures. Then, we evaluated the VM in xenograft tumors derived from MDA-MB-231, MCF7 and MDA-MB-361 using transmission electron microscopy (TEM). MDA-MB-231 xenografts displayed channel-like structures formed by tumor cells encompassing erytrocytes, whereas in MCF7 and MDA-MB-361 xenografts the endothelial lining delimiting blood vessels was clearly visible. Notably, blood vessels surrounded by tumor cells were also identified in human TN specimens processed for TEM, and these structures were significantly more frequent in TN compared to non-TN tumors. 60% reduction in TN vascular channel formation in vitro by an anti-bFGF monoclonal antibody along with no effect using anti-VEGF antibody indicated that TN breast carcinoma cells can generate vascular channels through bFGF-mediated pathway. Silencing of different receptors involved in bFGF signal (i.e. FGFR2 and PDGFR β) abrogated VM in TN cells. Finally, TNBC cells able to perform vascular-like channels were found to express significantly higher (p= 0.003) levels of FGFR related genes, described associated with basal-like BC aggressiveness, compared to all the other tested cell lines. Conclusions. Our findings point to the possibility that TNBC cells are maintained by proangiogenic signals and that increased sensitivity to Sunitinib probably relies on the specific impairment of PDGFRβ and FGFR2-mediated pathways, which might represent a possible specific therapeutic target. Partially supported by AIRC and Italian Bureau of Health. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-08-01.

HER2, member of HER family, is overexpressed in up to 30% of breast cancers and often associated to a more aggressive phenotype, resistance to chemotherapeutic agents and increased risk of metastasis. Anti-HER2 targeted therapies significantly increased the overall survival of HER2 breast cancer patients, however tumor cells frequently develop alternative resistance mechanisms and the molecular bases of this phenomenon are still far to be fully elucidated. A crucial issue is then represented by the urgent need of biomarkers able to predict the response to treatment, and certainly of new therapeutic tools to counteract the resistance mechanisms. The oncosuppressive role of miR-205 in the pathogenesis of breast cancer has been demonstrated, as well as a possible cross-regulation between miR-205 and HER signaling. MiR-205 is in fact negatively regulated by HER2 and is involved in the regulation of HER2-mediated proliferation by directly targeting HER3, preferential partner of HER2. We have investigated miR-205 role on trastuzumab resistance by using human HER2 overexpressing breast cancer cell lines and patient-derived xenograft (PDX) models. Here we show that HER3 silencing, either mediated by miR-205 or by a siRNA specific against HER3, is able to improve the responsiveness to Trastuzumab, reducing tumor cell growth and colony formation capability. This effect is mediated by impairment of Akt-mediated pathway.Combination of Trastuzumab treatment and in vivo delivery of miR-205-conjugated lipid nanoparticles in PDX models is currently on going. Finally, expression analysis of miR-205 in breast cancer patients treated in adjuvant with trastuzumab has provided new insights about its possible role as predictive biomarker of response. In summary, here we show that miR-205 improves the responsiveness to Trastuzumab treatment at least partially though HER3 silencing, and associated to outcome of patients treated in adjuvant setting, thus representing a potential adjuvant tool and predictive biomarker. Citation Format: Claudia Piovan, Elvira D’Ippolito, Ilaria Plantamura, Patrizia Casalini, Elda Tagliabue, Marilena V. Iorio. The promise of miR-205 in HER2+ breast cancer: predicting response to Trastuzumab and overcoming resistance. [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 1068.

It is established that the interaction between microenvironment and cancer cells has a critical role in tumor development, given the dependence of neoplastic cells on stromal support. However, how this communication promotes the activation of normal (NFs) into cancer-associated fibroblasts (CAFs) is still not well understood. Most microRNA (miRNA) studies focused on tumor cell, but there is increasing evidence of their involvement in reprogramming NFs into CAFs. Here we show that miR-9, upregulated in various breast cancer cell lines and identified as pro-metastatic miRNA, affects the properties of human breast fibroblasts, enhancing the switch to CAF phenotype, thus contributing to tumor growth. Expressed at higher levels in primary triple-negative breast CAFs versus NFs isolated from patients, miR-9 improves indeed migration and invasion capabilities when transfected in immortalized NFs; viceversa, these properties are strongly impaired in CAFs upon miR-9 inhibition. We also demonstrate that tumor-secreted miR-9 can be transferred via exosomes to recipient NFs and this uptake results in enhanced cell motility. Moreover, we observed that this miRNA is also secreted by fibroblasts and in turn able to alter tumor cell behavior, by modulating its direct target E-cadherin, and NFs themselves. Consistently with the biological effects observed, gene expression profiles of NFs upon transient transfection with miR-9 show the modulation of genes mainly involved in cell motility and extracellular matrix remodeling pathways. Finally, we were able to confirm the capability of NFs transiently transfected with miR-9 to promote in vivo tumor growth. Taken together, these data provide new insights into the role of miR-9 as an important player in the cross-talk between cancer cells and stroma.

Triple negative breast cancer (TNBC) represents a challenging tumor type due to their poor prognosis and limited treatment options. It is well recognize that clinical and molecular heterogeneity of TNBC is driven in part by post-transcriptional regulators such as miRNAs. To stratify TNBCs, we profiled 1050 miRNAs in 132 adjuvant TNBC tumors and 40 tumors from other immunophenotypes using an Affymetrix microarray platform. A NMF clustering analysis allowed us to identify 4 TNBC subtypes featuring unique miRNA expression patterns, disease free and overall survival rates and particular gene ontology enrichments (performed with GSEA algorithm). Our agglomerative approach was cross-validated by using two other clustering algorithms (k-means and consensus clustering). TNBC miRNAs subgroups were also correlated with the Lehmann intrinsic subtypes, finding a significant enrichment of immmnomodulartory and basal 1 subtypes in our high risk miRNA subgroups. 3 cell line models (MDA MB 468, MDA MB 231 and HS578T) were classified according to our miRNA signature, recapitulating two different miRNA subgroups. The TNBC tumors were compared against other phenotypes identifying differentially expressed miRNAs that together with the altered miRNAs within the subgroups allowed us to define interesting miRNAs for further functional analysis. We found low expression levels of miR-342-3p in TNBC tumors compared with other breast cancer phenotypes, and this down-regulation characterizes one of our miRNA subgroups with high risk to relapse. To characterize the functional its functional role, miR-342-3p was transiently transfected in the cell line MDA-MB-468, showing a decrease in cell proliferation, viability and migration rates. A gene expression profile (Human gene st, Affymetrix) revealed 140 altered mRNAs, from which 35 are potential direct targets of miR-342-3p defined by an in-silico analysis. The monocarboxylate transporter 1(MCT1), was confirmed as one target of miR-342-3p by a luciferase assay and western blot analysis. MCT1 repression by the miRNA promotes lactate efflux changes in the tumor cells, reflected in the accumulation of exogenous lactate and the increase in levels of extracellular endogenous lactate together with a decrease level of intra and inter cellular glucose concentration. We also explored other mechanisms that can contribute to the modulation of lactate and glucose levels such as the expression of MCT4, HIF1alpha and Glut1, but not significant changes were observed, pointing out the important role of the regulatory circuit of miR-342-3p and MCT1 in a particular TNBC tumor cells metabolism. These data suggest a metabolic change that favors a more glycolytic environment, which lead to a glucose deprivation context that may contribute to the reduction in proliferation, viability and migration capabilities already described. Furthermore, to define the main transcriptional modulator of the intronic miR-342-3p in TNBC, we evaluated the correlation between the expression of estrogen receptor, miR-342-3p and its host gene EVL in TNBC tumors from TCGA data. A significant correlation between these transcripts was observed. These results, together with information from other studies and Chip-seq analysis suggest that the ER activates the transcription of EVL and consequently the intronic miR-342-3p. We validated these correlations in our cellular model by transiently silencing of the ER. Our data provides evidence that miRNAs sub-classification signature can recapitulate the biological and clinical heterogeneity of TNBC. Moreover, show that miRNAs regulate important oncogenic pathways in TNBC cells such as: proliferation, cell movement and apoptosis in the different subgroups. Citation Format: Sandra L. Romero-Cordoba, Sergio Rodriguez-Cuevas, Rosa Rebollar-Vega, Veronica Bautista-Pina, Antonio Maffuz-Aziz, Elda Tagliabue, Marilena Iorio, Elvira D'Ippolito, Sara Baroni, Ilaria Plantamura, Alfredo Hidalgo-Miranda. A microRNA signature identifies subtypes of triple-negative breast cancer and reveals miR-342-3p as regulator of a lactate metabolic pathway through silencing monocarboxylate transporter 1. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A47.

Tumor vascularization is a fundamental step in solid tumor progression and is orchestrated by different pathways of vasculogenesis. In malignant tumors, neoplastic cells can differentiate into endothelial-like cells acquiring the expression of endothelial markers (i.e. CD31 and CD34) and participating in the formation of vascular-like structures that functionally deliver oxygen and nutrients to the tumor site. We recently identified PDGFRβ as an important player of this process in triple negative breast cancer (TNBC). Interestingly, increasing evidence supported a connection between PDGFRβ and epithelial to mesenchymal transition (EMT), important step for the endothelial trans-differentiation process. PDGFR signaling arose as a promising target for TNBC; thus a better understanding of this new role of PDGFRβ could be relevant in the biology and treatment of TNBC. We then aimed at investigating microRNAs able to regulate tumor vascularization via PDGFRβ-mediated endothelial differentiation in TNBC, focusing on miR-9 and miR-200 family in light of their known relation with PDGFRβ and EMT. Tube formation assay showed that in MDA-MB-231 and MDA-MB-157 TNBC cell lines transfection of miR-9 and miR-200 family members (miR-200b and miR-200c) promoted or inhibited, respectively, the ability of cells to form vascular-like structures when seeded on matrigel. Stimulation of PDGFRβ with PDGF-BB ligand induced miR-9 expression and enhanced the loop formation ability of treated cells. This advantage was, however, almost completely abrogated by the concomitant inhibition of miR-9, thus demonstrating that miR-9 contributed to PDGFRβ-mediated vasculogenic properties of TNBC. Moreover, we found that silencing of STARD13, direct target of miR-9 validated by luciferase reporter assay, improved the vasculogenic potential. Ectopic expression of miR-200 members, instead, suppressed PDGFRβ at protein level in both TNBC cell lines. Furthermore, the silencing of ZEB1, known target of miR-200 family, induced downregulation of PDGFRβ at protein and mRNA level; interestingly, the mRNA levels of ZEB1 and PDGFRβ strongly correlated in the TNBC subset of the TGCA dataset. The in vivo effect of miR-9 and miR-200 family on vasculogenic properties of TNBC was then validated first through the generation of MDA-MB-231 clones for stable inhibition of miR-9 and restoration of miR-200c; the resulting miR-9 sponge and miR-200c xenografted tumors showed lower number of vascular lacunae than controls, identified through immunohistochemical (IHC) staining for CD31. These results were validated in orthotopic MDA-MB-231 breast tumors treated with miR-9 inhibitors or miR-200c mimics by peritumoral injection. We finally evaluated PDGFRβ, by IHC, and miR-9 and miR-200c, by Real-time PCR, in a cohort of TNBC. IHC analysis demonstrated that PDGFRβ staining identified tumor cells participating in vascular lacunae, data further confirmed by immunofluorescence co-localization of PDGFRβ and CD31. Interestingly, unlike miR-9, the expression of miR-200c negatively associated with both the presence of vascular lacunae and tumor nests positive for PDGFRβ. Finally, in situ hybridization analysis revealed that miR-200c was mainly expressed by tumor cells. In conclusion, our results suggest that miR-9 and miR-200 family play an opposite role in the regulation of the vasculogenic aptitude of TNBC. MiR-9 is induced by PDGFRβ and enhances the vasculogenic properties of tumor cells in part through the negative regulation of STARD13. MiR-200, instead, inhibits this aggressive phenotype indirectly suppressing PDGFRβ through targeting of ZEB1. Citation Format: Elvira D'Ippolito, Ilaria Plantamura, Lucia Bongiovanni, Patrizia Casalini, Sara Baroni, Claudia Piovan, Rosaria Orlandi, Ambra V. Gualeni, Annunziata Gloghini, Anna Rossini, Filippo De Braud, Elda Tagliabue, Claudio Tripodo, Marilena V. Iorio. miR-9 and miR-200 regulate PDGFRβ-mediated endothelial differentiation of neoplastic cells in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A18.

An increasing body of evidence highlights an intriguing interaction between microRNAs and transcriptional factors involved in determining cell fate, including the well known “genome guardian” p53. Here we show that miR-205, oncosuppressive microRNA lost in breast cancer, is directly transactivated by oncosuppressor p53. Moreover, evaluating miR-205 expression in a panel of cell lines belonging to the highly aggressive triple negative breast cancer (TNBC) subtype, which still lacks an effective targeted therapy and characterized by an extremely undifferentiated and mesenchymal phenotype, we demonstrated that this microRNA is critically down-expressed compared to a normal-like cell line. Re-expression of miR-205 where absent strongly reduces cell proliferation, cell cycle progression and clonogenic potential in vitro, and inhibits tumor growth in vivo, and this tumor suppressor activity is at least partially exerted through targeting of E2F1, master regulator of cell cycle progression, and LAMC1, component of extracellular matrix involved in cell adhesion, proliferation and migration.

Despite the improvement in the management of HER2+ breast cancer patients with the approval of Trastuzumab in the clinical practise, and the success rate of this treatment, a still considerable percentage of patients do not respond or eventually develop a secondary resistance, leading to relapse and poor outcome. A crucial issue is represented by the urgent need of biomarkers able to predict the response to treatment, and certainly of new therapeutic tools to counteract the resistance mechanisms. It is known that resistance to anti-HER2 therapies can be due to escape mechanisms triggered by alternative receptors: here we show that HER3 silencing, either mediated by miR-205, known to directly target the receptor (Iorio MV et al., 2009) or by a siRNA specific against HER3, is able to improve the responsiveness to Trastuzumab, reducing tumor cell growth and colony formation capability. This effect seems to be mediated by impairment of the Akt-mediated pathway. Finally, evaluating miR-205 expression in a series of 51 HER2+ breast cancer patients treated with Trastuzumab in adjuvant setting, we observed that miR-205 is significantly associated with outcome: setting the median expression as cut-off, and performing a contingency analysis (Fisher's exact test), miR-205 expression was above the median in a significantly (p=0.03) higher percentage of responders versus non responders patients (61% versus 26%). In summary, here we show that miR-205 improves the responsiveness to Trastuzumab treatment at least partially though HER3 silencing, and associated to outcome of patients treated in adjuvant setting, thus representing a potential adjuvant tool and predictive biomarker. Citation Format: Elvira D'Ippolito, Claudia Piovan, Ilaria Plantamura, Marta Giussani, Patrizia Casalini, Manuela Campiglio, Elda Tagliabue, Marilena V. Iorio. MiR-205 and Trastuzumab: Potential as adjuvant therapeutic tool and predictive biomarker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4381. doi:10.1158/1538-7445.AM2014-4381

Triple negative breast cancers (TNBC) account for 15% of breast cancers. TNBCs carry a high risk of recurrence and deaths, due to the high rate of local and systemic relapse in these patients and no therapeutic options except chemotherapy are currently available. The TNBC pattern of recurrence present a distant recurrence peak at approximately 3 years and then declines rapidly thereafter, whereas in all non-TNBC types the recurrence risk seems to be constant over time. TNBC relapse risk is comparable to that of HER2−positive tumors subtype, in which growth-factors released during the healing process accelerate the early recurrences in HER2−positive patients. Thus, we speculate that also TNBC early relapse may depend on their capability to respond to wound-healing stimulation. To this aim, TNBC were treated with drainages to identify which receptors/pathways can be activated and play a driving role in TNBC progression. A pilot reverse phase protein microarray (RPMA) experiment on MDA-MB-231 TN cells drainage-fluids stimulated revealed a specific activation of PDGFR and VEGFR and their downstream pathways, whereas no significant changes were observed in other receptors, such as EGFR, IRS, Met and ERB3. The type of activated receptors suggested the involvement of endothelial receptors upon drainages stimulation and, indeed TNBC cell lines expressed endothelial molecules, such as CD34, CD31, CD146. Beside the proved role of some of these receptors in cellular proliferation, the TNBC endothelial-like phenotype prompt us to analyzed TNBC cell lines capability to form vascular-like channels when seeded on matrigel. Drainages were able to accelerate the formation of vascular channels in TNBC cell lines and, moreover to consistently increase proliferation of TNBC cells compared to non-TNBC cells. To prove whether receptors found activated by drainages play a key role in TNBC progression, we targeted PDGFR, VEGFR and other receptors possibly involved in angiogenesis and vasculogenic mimicry with sunitinib (targeting PDGFR, VEGFR, FGF and c-kit), anti-bFGF antibody (Ab)(targeting the ligand bFGF) and bevacizumab (targeting the VEGF) in TNBC cells drainage-stimulated in vitro. Sunitinib and anti-bFGF Ab halved the proliferation of TNBC cell lines and reduced of almost 60% the formation of vascular-like channels in TNBC cells, whereas bevacizumab modestly affect proliferation but not vasculogenic properties. Notably, sunitinib and anti-bFGF Ab strongly inhibited MDA-MB-231 and MDA-MB-468 xenografts tumor growth (sunitinib: 80%, and 70% Growth Index (GI), respectively; anti-bFGF Ab 70% and 60% GI, respectively) whereas bevacizumab determined no more than 30% decrease of tumor volume. Unfortunately, all these drugs did not efficiently control the development of lung metastases, that indeed significant increased compared to their control, possibly through induction of hypoxia processes. In conclusion, wound healing promotes TNBC progression by sustaining proliferation and vasculogenesis. The use of sunitinib and anti-bFGF antibody strongly inhibited tumor growth in mice models, but significantly increased lung metastases suggesting a combined use of these drugs with molecules able to interfere with hypoxia pathway. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-07.

An increasing number of experimental evidences show that microRNAs can have a causal role in breast cancer tumorigenesis as a novel class of oncogenes or tumor suppressor genes, depending on the targets they regulate. miR-205, down-modulated in breast cancer versus normal breast tissue, was originally associated to absence of vascular invasion and shown to be involved in the EMT. Moreover, we recently demonstrated that miR-205 directly targets HER3 receptor and impairs the Akt-mediated survival pathway, thus inhibiting clonogenic potential of SKBr3 cells and increasing the responsiveness to tyrosin-kinase inhibitors. Considering the striking evidences of a major role of miR-205 as oncosuppressor in breast cancer, we decided to investigate its possible involvement in another tumor subgroup, extremely aggressive and still lacking a specific therapy: triple negative breast tumors. Here we show that re-introduction of miR-205 in the triple negative breast cancer cell line MDA-MB-231 led to a dramatic reduction of both proliferation and clonogenic potential as well as migration capability. Notably, miR-205 also significantly inhibited the in vivo growth in xenograft models obtained injecting stable clones of MDA-MB-231 cells transfected with a miR-205-encoding plasmid or the empty vector. Investigating alternative pathways potentially regulated by miR-205 in this model, we noticed that many of the predicted targets are actually interconnected and related to adhesion and migration processes. Indeed, this microRNA seems to be involved in the control of cell-cell and cell-matrix adhesion regulating the expression of extracellular matrix-related molecules. Among them, we focused on LAMC1, reported to be involved in cell adhesion, differentiation, migration, signaling and metastasis. By Western Blot and Luciferase assay, we demonstrated that miR-205 directly targets LAMC1 in MDA-MB-231 cells. In conclusion, miR-205 exerts a role as oncosuppressor gene also in triple negative breast cancer: it exerts a dramatic inhibition of proliferation and migration in triple negative MDA-MB-231 cell line, at least partially through direct regulation of LAMC1. These evidences not only underline the consistent role of miR-205 in the biology of different breast cancer subgroups, but provide the rational bases for a therapeutic intervention miRs-based. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2089.

Triple negative (TN) breast cancers account for about 15-20% of breast cancers and are associated with aggressiveness, poor prognosis and shorter survival. Because of the lack of specific therapy guidelines for this subgroup there is an urgent need to define specific markers for therapeutic targeting in these tumors. We observed that TN breast cancer cells lines are able to perform “Vasculogenic Mimicry” (VM) in vitro. In fact, all four TN cell lines evaluated (MDA-MB-231, MDA-MB-157, MDA-MB-468 and HCC1937) were able to form vascular channels when seeded on the murine tumor-derived basement membrane (Matrigel), whereas luminal (MCF7 and ZR-75-1) and HER2-positive (MDA-MB-361 and SKBr3) breast carcinoma cell lines did not exhibit vascular structures. Then, we evaluated the VM in xenograft tumors derived from MDA-MB-231, MCF7 and MDA-MB-361 using transmission electron microscopy (TEM). In MDA-MB-231 xenografts it has been revealed channel-like structures formed by tumor cells encompassing erytrocytes, whereas in MCF7 and MDA-MB-361 xenografts the endothelial lining delimiting blood vessels was clearly visible. Notably, blood vessels surrounded by tumor cells were also identified in human TN specimens processed for TEM, and these structures were significantly more frequent in TN compared to non-TN tumors. 60% reduction in TN vascular channel formation in vitro by an anti-bFGF monoclonal antibody together with no effect using anti-VEGF antibody indicated that TN breast carcinoma cells can generate vascular channels through bFGF-mediated pathway. Silencing different receptors involved in bFGF signal (i.e. FGFR2 and PDFGR) abrogated VM in TN cells. Treatment of MDA-MB-231 xenografts with a TKI able to block both FGFR and PDGFR induced tumor regression, versus only slight growth inhibition in tumors derived from MCF7 luminal cell line. In conclusion our findings point to the possibility that TN tumors generate vascular channels independent of tumor angiogenesis and that targeting this VM capability may represent a valid therapeutic tool. (Partially supported by AIRC). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1598.