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Background: Despite great efforts to identify tumor-related prognostic biomarkers in early-stage Human Epidermal growth factor Receptor 2 (HER2)-positive Breast Cancer (HER2+ BC), reliable determinants of long-term clinical outcomes are lacking. HER2+ BC is a lipogenic malignancy, and HER2-driven fatty acid (FA) biosynthesis plays a crucial role in sustaining cancer cell growth, proliferation, and metastatization. Recently, FA uptake, a process mediated by the transmembrane transporter CD36, has emerged as a new determinant of acquired resistance to anti-HER2 treatments in preclinical studies. Methods: The phase III NeoALTTO (NCT00553358) trial randomized 455 HER2+ BC patients to receive lapatinib, trastuzumab, or the combination of both drugs with weekly paclitaxel, followed by surgery, adjuvant fluorouracil, epirubicin, and cyclophosphamide and the same anti-HER2 therapy received before surgery. In the present analysis, we tested the hypothesis that higher intratumor CD36 mRNA levels could be associated with worse Event Free Survival (EFS) in a cohort of NeoALTTO patients for whom global gene expression analysis of baseline tumor samples through ClariomS platform was available. Results: A total of 180 patients were included (trastuzumab arm: n=66; lapatinib arm: n=65; combination arm: n=49). Higher baseline CD36 expression levels were associated with significantly worse EFS in patients treated with trastuzumab-based therapy, at both univariate (continuous scale, HR:1.73, 95% CI 1.20-2.49) and multivariable (HR:1.72, 95% CI 1.20-2.46) analysis, but not in patients treated with lapatinib- or trastuzumab plus lapatinib (HR:1.01; 95% CI: 0.69-1.47; p=0.98 and HR:1.02; 95% CI:0.54-1.95; p=0.94, respectively). CD36 levels did not predict pathological Complete Response (pCR) probability, while combining CD36 expression (based on median levels) with pCR status identified two subsets of patients with especially bad or good prognosis (7-year EFS rates in patients with high CD36/no pCR: 47%, 95% CIs: 0.26-0.66 vs. 79%, 95% CIs: 0.62-0.88 in patients with low CD36/any pCR status (Yes/No) OR high CD36/pCR; Log-Rank test p= 0.006). Conclusions: This is the first study to show that high CD36 expression is independently predictive of worse long-term clinical outcomes in HER2+ BC patients treated with neoadjuvant trastuzumab-based therapy. Along with recent preclinical data highlighting CD36 role in tumor resistance to anti-HER2 therapies, our findings point to CD36 as a novel promising target for HER2+ BC treatment. Citation Format: Francesca Ligorio, Serena Di Cosimo, Paolo Verderio, Chiara Maura Ciniselli, Sara Pizzamiglio, Lorenzo Castagnoli, Tiziana Triulzi, Elda Tagliabue, Sarra El-Abed, Miguel Izquierdo, Evandro de Azambuja, Paolo Nuciforo, Jens Huober, Luca Moscetti, Wolfgang Janni, M Coccia-Portugal, Paola Corsetto, Antonino Belfiore, Daniele Lorenzini, Maria Grazia Daidone, Andrea VIngiani, Serenella M Pupa, Giancarlo Pruneri, Claudio Vernieri. High CD36 expression predicts worse event free survival in HER2-positive breast cancer patients treated with neoadjuvant trastuzumab-based therapy: An exploratory analysis of 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 P2-13-12.

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.

Cancer is a systemic disease able to reprogram the bone marrow (BM) niche towards a protumorigenic state. The impact of cancer on specific BM subpopulations can qualitatively differ according to the signals released by the tumor, which can vary on the basis of the tissue of origin. Using a spontaneous model of mammary carcinoma, we identified BM mesenchymal stem cells (MSC) as the first sensors of distal cancer cells and key mediators of BM reprogramming. Through the release of IL1B, BM MSCs induced transcriptional upregulation and nuclear translocation of the activating transcription factor 3 (ATF3) in hematopoietic stem cells. ATF3 in turn promoted the formation of myeloid progenitor clusters and sustained myeloid cell differentiation. Deletion of Atf3 specifically in the myeloid compartment reduced circulating monocytes and blocked their differentiation into tumor-associated macrophages. In the peripheral blood, the association of ATF3 expression in CD14+ mononuclear cells with the expansion CD11b+ population was able to discriminate between women with malignant or benign conditions at early diagnosis. Overall, this study identifies the IL1B/ATF3 signaling pathway in the BM as a functional step toward the establishment of a tumor-promoting emergency myelopoiesis, suggesting that ATF3 could be tested in a clinical setting as a circulating marker of early transformation and offering the rationale for testing the therapeutic benefits of IL1B inhibition in patients with breast cancer. Significance: Bone marrow mesenchymal stem cells respond to early breast tumorigenesis by upregulating IL1B to promote ATF3 expression in hematopoietic stem cells and to induce myeloid cell differentiation that supports tumor development.

Although the clinical benefit of trastuzumab for the management of HER2-positive breast carcinomas (BCs) has been largely demonstrated, many women, even those reported to have tumors potentially most sensitive to trastuzumab (e.g., HER2-enriched by PAM50 and with infiltrating immune cells) do not respond to this agent. The relevance of immunity in the cytotoxic mechanism of action of trastuzumab supports the notion that the anti-tumor effect of this monoclonal antibody depends on host immune system activity. Since gut commensal bacteria reportedly contribute to the development and maintenance of the immune system and have immunomodulatory effects, we investigated whether a relationship between gut microbiota composition and the response to trastuzumab exists in patients with HER2-positive BC. Stool samples were collected from 18 patients with primary HER2-positive BCs before the outset of neoadjuvant trastuzumab-based chemotherapy and analyzed by 16S rRNA gene profiling using Illumina Miseq platform. Gut microbiota β-diversity analysis by UniFrac algorithm revealed a heterogeneous microbiota composition mainly due to differences in the relative abundance of Bacteroides, Faecalibacterium and a genus belonging to Ruminococcaceae family. Unsupervised analysis identified two microbiota clusters that significantly discriminated patients according to pathological complete response (pCR) (p=0.0128, by Fisher test). No association between microbiota clusters and PAM50 molecular classification of tumor biopsies, as evaluated by gene expression, was observed. Moreover, HER2-enriched cases were distributed in the two microbiota clusters based on trastuzumab response. Differences in the intestinal microbiota between responsive (R) and non-responsive (NR) patients were assessed by LEfSe analysis: a significant higher and lower abundance of Clostridiales taxonomic order and Bacteroides taxonomic genus, respectively, was foundin R as compared to NR patients. Immune genes expressed in tumor biopsies that were found significantly correlated with these bacteria mainly belong to innate and adaptive immune response and cellular response to tumor necrosis factor pathways. To investigate the causal role of gut microbiota in trastuzumab benefit, FVB mice bearing syngeneic mammary carcinoma overexpressing human HER2 were transplanted with fecal material from R and NR patients after intestinal flora depletion by the use of an antibiotic cocktail. The mouse response to trastuzumab treatment recapitulated the response observed in patients from which stool derived. The obtained results support the contribution of gut microbiota in trastuzumab activity by influencing tumor immune microenvironment, independently of tumor intrinsic molecular characteristics. These data represent the proof-of-concept that manipulating gut microbes in resistant patients could be a new strategy to improve the response to trastuzumab. Supported by AIRC Citation Format: Elda Tagliabue, Martina Di Modica, Giorgio Gargari, Viola Regondi, Arianna Bonizzi, Stefania Arioli, Fabio Corsi, Simone Guglielmetti, Tiziana Triulzi. Commensal gut microbiota influences efficacy of trastuzumab in patients with HER2-positive breast carcinoma [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-32.

Emerging evidence indicates that gut microbiota affect the response to anticancer therapies by modulating the host immune system. In this study, we investigated the impact of gut microbiota on immune-mediated trastuzumab antitumor efficacy in preclinical models of HER2-positive breast cancer and in 24 patients with primary HER2-positive breast cancer undergoing trastuzumab-containing neoadjuvant treatment. In mice, the antitumor activity of trastuzumab was impaired by antibiotic administration or fecal microbiota transplantation from antibiotic-treated donors. Modulation of the intestinal microbiota was reflected in tumors by impaired recruitment of CD4+ T cells and granzyme B–positive cells after trastuzumab treatment. Antibiotics caused reductions in dendritic cell (DC) activation and the release of IL12p70 upon trastuzumab treatment, a mechanism that was necessary for trastuzumab effectiveness in our model. In patients, lower α-diversity and lower abundance of Lachnospiraceae, Turicibacteraceae, Bifidobacteriaceae, and Prevotellaceae characterized nonresponsive patients (NR) compared with those who achieved pathologic complete response (R), similar to antibiotic-treated mice. The transfer of fecal microbiota from R and NR into mice bearing HER2-positive breast cancer recapitulated the response to trastuzumab observed in patients. Fecal microbiota β-diversity segregated patients according to response and positively correlated with immune signature related to interferon (IFN) and NO2-IL12 as well as activated CD4+ T cells and activated DCs in tumors. Overall, our data reveal the direct involvement of the gut microbiota in trastuzumab efficacy, suggesting that manipulation of the gut microbiota is an optimal future strategy to achieve a therapeutic effect or to exploit its potential as a biomarker for treatment response. Significance: Evidence of gut microbiota involvement in trastuzumab efficacy represents the foundation for new therapeutic strategies aimed at manipulating commensal bacteria to improve response in trastuzumab-resistant patients. See related commentary by Sharma, p. 1937

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.

BACKGROUND: As a neoadjuvant regimen for HER2-positive early breast cancer (BC), the use of two HER2-directed agents is more effective in producing pathological complete (pCR) responses than trastuzumab alone. Nevertheless, highly effective dual anti-HER2 combination may be unnecessary in patients who already benefit from a single agent. We previously reported that our 41-gene TRAR score is an accurate predictor of response to trastuzumab, with low scores being predictive of response to trastuzumab and favorable prognosis (Triulzi T. et al., 2015). PATIENTS AND METHODS: Tissue specimens from HER2-positive BC patients of Neo-ALTTO trial who received neoadjuvant trastuzumab and/or lapatinib plus paclitaxel were included in this study. Analysing RNA from fresh tissue using the 41-gene signature test, the area under the ROC curve (AUC) and the corresponding 95% confidence interval was computed to evaluate the predictive ability of TRAR score with respect to pCR, the primary endpoint of Neo-ALTTO. The prognostic role of TRAR score was investigated using a Cox regression model in univariate fashion. The patterns of Event Free Survival (EFS) according to the dichotomized TRAR score were estimated using the Kaplan–Meier method. RESULTS: The TRAR score was assessed for 226 of the 455 (49.7%) patients enrolled in the Neo-ALTTO study: 136 (60%) presented with T2 tumors, 188 (83%) with N0/1 and 128 (56.6%) with estrogen receptor negative BC. In details, basal TRAR score was available for 69, 79 and 78 patients assigned to neoadjuvant trastuzumab, lapatinib, and their combination, respectively. Overall, patients achieving a pCR showed significantly lower levels of TRAR score than those with residual disease (p CONCLUSION: Overall, we show that our 41-gene signature is accurate in predicting patient response to neoadjuvant HER2 targeted therapy in terms of pCR. In particular, low levels of TRAR score can identify a HER2-positive breast cancer subgroup highly responsive to trastuzumab as monotherapy for whom combination with other HER2-targeted drugs does not appear justified and may be one tool used for exploring de-escalating strategies without sacrificing outcomes. Citation Format: Di Cosimo S, Triulzi T, De Cecco L, Pizzamiglio S, de Azambuja E, Fumagalli D, Pusztai L, Harbeck N, Izquierdo M, de la Pena L, Huober J, Baselga J, Piccart M, Verderio P, Tagliabue E. Identifying clinically relevant subgroups of women with HER2-positive breast cancer: An analysis of Neo-ALTTO using the 41-gene TRAR score [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-03.

It is well know that trastuzumab activity against HER2-positive breast carcinomas (BCs) depends also on immune cells. Our genomic analysis of 53 HER2-positive BCs indicated that those exquisitely sensitive to treatment are addicted to this oncoprotein and are enriched in immune pathways and T-cells infiltration, raising the hypothesis that HER2 regulates immune cells recruitment. The aim of this study was to investigate how HER2 activity contributes to mold tumor microenvironment. Gene expression profile analysis of 53 HER2-pos BCs showed that trastuzumab-sensitive tumors express significantly higher levels of chemokines involved in immune cells recruitment (IFNγ-related CXCLs and CCLs) and higher levels of immune checkpoint ligands (PD-L1, PD-L2) than tumors that do not benefit from trastuzumab. Immunohistochemistry analysis on FFPE tumor specimens showed a significant positive association between tumor cell positivity for CXCL9, CXCL10 and CCL2 and trastuzumab sensitivity. In vitro analysis in HER2-positive BC cell lines revealed that while IFNγ-related chemokines (CXCL9 and CXCL10) are not directly regulated by HER2 signaling, CCL2 was significantly up-regulated both at mRNA and protein levels by HER2 stimulation with EGF/HRG and down-modulated by HER2 inhibition with trastuzumab. The EGF-induced increase in CCL2 expression was abrogated by the concomitant treatment with PI3K inhibitor but not with MEK inhibitor, indicating that HER2 regulates CCL2 expression via the PI3K pathway. HER2 signals derived from cell stimulation with EGF/HRG caused also the upmodulation of PD-L1/PD-L2 both at mRNA and protein levels while their inhibition with trastuzumab reduced their levels. Overall, our results support the notion that the activated HER2 oncogene concomitantly induces the expression of CCL2 and PD-L1/PD-L2 regulating infiltration of pro-trastuzumab immune cells and their suppression. Thus, it is likely that activity of trastuzumab in therapy-responsive tumors derives from its ability in relieving suppression of recruited antitumor effector immunity. Supported by Italian Association for Cancer Research (AIRC). Citation Format: Tagliabue E, Forte L, Regondi V, Ghirelli C, Aiello P, Triulzi T. HER2 activity regulates the pro-trastuzumab immune tumor microenvironment [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-21-33.

Background: Although anti-HER2 treatments, including the monoclonal antibodies trastuzumab (T) and pertuzumab (P) and the tyrosine kinase inhibitor lapatinib (L), have remarkably improved the prognosis of patients with HER2-positive breast cancer (HER2+ BC), acquired resistance to these compounds remains a major clinical issue. HER2-mediated signaling induces fatty acid (FA) de novo biosynthesis, and ongoing clinical trials are investigating FA synthase enzyme (FASN) inhibitors in combination with anti-HER2 treatments. We hypothesized that by inhibiting FA biosynthesis, HER2 inhibitors make HER2+ BC cells dependent on extracellular FA uptake, which could contribute to resistance to anti-HER2 treatments. Methods: In tumor biopsy specimens from HER2+ BC patients treated with preoperative T-containing chemotherapy (ChT), we performed quantitative real time PCR (qRT-PCR) to measure expression levels of the lipoprotein lipase (LPL) enzyme and the FA transporter CD36, two key players in the uptake of extracellular FAs. The same analyses were also performed in surgical tumor specimens from patients failing to achieve pathologic complete response (pCR), and paired Wilcoxon test was used to compare CD36 expression levels of pre- versus post-treatment tumors. In MDA-MB-361, HCC1954 and BT474 HER2+ BC cell lines, we investigated the association between baseline CD36 expression and cells’ sensitivity to L, or their ability to uptake the docosahexanoic acid (DHA) from culture media. Finally, we assessed the antiproliferative effect of combining L with the CD36 inhibitor Sulfo N-Succinimidoyl Oleate (SSO). Results: 82 patients with stage II-III HER2+ BC were treated with preoperative ChT plus T at Fondazione IRCCS Istituto Nazionale dei Tumori between January 2013 and December 2017. As expected, achieving pCR was associated with better relapse-free survival (RFS) and overall survival (OS). Baseline LPL and CD36 expression did not correlate with pCR probability (p=0.15 and 0.1, respectively), nor with RFS (p=0.28 and 0.7, respectively) or OS (p=0.42 and 0.35, respectively). On the other hand, in 20 patients failing to achieve pCR, we found significantly increased LPL (p=0.00096) and a trend towards increased CD36 (p=0.12) expression in post-treatment surgical specimens. In a parallel cohort of 17 stage II-III HER2+ BC patients enrolled in a prospective study, LPL and CD36 expression was significantly increased 21 days after the first administration of single-agent T (p=0.0002 and p Citation Format: Claudio Vernieri, Lorenzo Castagnoli, Francesca Ligorio, Antonino Belfiore, Elena Fasano, Paola A. Corsetto, Simona Faraci, Marta Brambilla, Francesca Corti, Tiziana Triulzi, Daniele Generali, Andrea Vingiani, Angela M. Rizzo, Giulia V. Bianchi, Giuseppe Capri, Elda Tagliabue, Filippo de Braud, Giancarlo Pruneri, Serenella M. Pupa. Fatty acid uptake as a potentially new resistance mechanism to anti-HER2 treatments in HER2-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-01.

BACKGROUND AIs are the milestone adjuvant treatment for postmenopausal early HR+ BC pts. Non-steroidal AIs induce a significant oestrogen deprivation, responsible in approximately 40% of the pts for muscle-skeletal adverse events (MSKs) and for discontinuation in 25%. The present study aims to evaluate the impact of the diagnosis communication and of the need for AI therapy on the future development of MSKs events in a prospective cohort of EBC pts. PATIENTS AND METHODS The analysis of patients’ psychological attitude was conducted by using 3 different tests: SCL 90-r for psychological symptoms, SF-36 for the self-perception of the health status and COPE-NVI for evaluation of coping. After a full explanation of the AIs potential adverse events by the oncologist, the questionnaires were self-administered with the support of a dedicated psychologist at the moment of the first visit (T0). For the future correlation between baseline psychological attitude and the development of AI-related events, the presence and the intensity of pain were measured by NRS 11-points scale and by Tender Points (TPs) evaluation. Pearson correlation analysis was used to calculate correlations between Pain Scale, Physical Synthetic Index of Sf-36 and NRS/TRPs scores. Wilcoxon’s Test was used to compare baseline scores with subsequent ones. RESULTS From October 2012 to April 2013, 70 EBC pts entered the study. Exclusion criteria included severe osteoporosis or other co morbidities that contraindicate therapy with AIs and documented diagnosis of psychosis or cognitive deterioration. Median age was 64.8 years (43-88). The vast majority of the pts (584.3%) presented stage IA-IIA disease. Fifteen pts aged less than 55 years were menopausal, 13 have previously received chemotherapy as part of the planned adjuvant programme. Thirty-four pts (48.6%) had a baseline NRS>0 and 18 NRS>5; 26 pts presented at least 1 positive TP. Regarding the SCL 90-r analysis, all the pts have reported standardized scores within the normal ranges for somatization, anxiety and phobic anxiety. The analysis of SF-36 questionnaires indicated that pts showed a significant lower score in comparison to the reference population in the ISF Index (44.17 vs 48.7) and in the Physical Health Scale (47.5 vs 74.13). These differences could suggest a greater difficulty to do daily activities and a worse global index of self physical perception At baseline, there was a statistically significant correlation between Physical Pain Scale of SF-36 and NRS Pain score (p=0.001) and between Physical Synthetic Index of SF-36 and NRS Pain Score (p CONCLUSION Early BC pts who are candidate for endocrine adjuvant therapy with AIs often present an impairment in terms of physical activity (daily and working) and perception of physical pain. The analysis of these parameters along the 1st year of AI therapy and their correlation with MSK adverse events onset is ongoing. Citation Format: Marina E Cazzaniga, Annalisa Valentini, Francesca Gallina, Francesca Riva, Alessandra Crippa, Marco Tagliabue, Federica Cicchiello, Diego Cortinovis, Paolo Bidoli. Psychological status of early breast cancer (EBC) patients (pts) after surgery and before the starting of aromatase Inhibitors (AIs). Results of a single-institution, prospective study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-12-05.

While results thus far demonstrate the clinical benefit of trastuzumab in breast cancer, some patients do not respond to this reagent. Identifying a robust clinical or molecular predictor of adjuvant trastuzumab benefit has proven challenging and even the most obvious candidate biomarker for a predictor of trastuzumab benefit, HER2 expression as assessed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), has proven to be surprisingly ambiguous in realizing benefit from the antibody. Results of a recent study that profiled expression of select genes in archived formalin-fixed, paraffin-embedded (FFPE) tumor blocks from a randomized study of breast cancer patients treated with adjuvant trastuzumab support a relationship between ERBB2/ESR1 mRNA levels and trastuzumab sensitivity. Based on that observation, we conducted whole-genome profiling by DASL technology of archival FFPE tumor blocks from 53 HercepTest 3+/2+ FISH-positive patients treated with adjuvant trastuzumab in our Institute; 308 genes were significantly associated with relapse-free survival by Cox proportional hazard model (α Together, our findings strongly suggest that ERBB2 mRNA, but not protein levels, mirror HER2 activity and thus oncogene addiction of tumor cells and susceptibility to trastuzumab in amplified HER2-positive tumors. Supported by AIRC. Citation Format: Elda Tagliabue, Viola Regondi, Loris De Cecco, Serenella M Pupa, Manuela Campiglio, Maria Luisa Carcangiu, Sylvie Ménard, Tiziana Triulzi. Correlation between ERBB2 mRNA levels, HER2-dependence and susceptibility to trastuzumab in human breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-15-06.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with high recurrences and mortality rate, for which no therapies besides chemotherapy are available to date. Lacking specific markers for an effective targeted therapy, TNBCs continue to represent the most important challenge for clinical oncologists. Here, we investigated the expression of CDCP1, a transmembrane non-catalytic receptor reportedly associated with poor prognosis in some solid tumors (e.g., lung and pancreatic cancer), and its association with tumor aggressiveness in a cohort of 115 human TNBC primary specimens obtained from women surgically treated in our Institute from the beginning of 2002 to the end of 2006 and selected based on immunohistochemical (IHC) criteria ( Together, our results strongly suggest that CDCP1 is a marker of aggressiveness able to identify cases with poorer prognosis among N-positive TNBCs and, noticeably, overexpression of CDCP1 in human primary TNBCs can reflect a CDCP1 genetic gain. Supported by AIRC. Citation Format: Tagliabue E, Turdo F, Bianchi F, Sandri M, Forte L, Casalini P, Gasparini P, Agresti R, Triulzi T, Sozzi G, Campiglio M. CDCP1 as a new marker of aggressiveness in triple-negative breast cancers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-11.

Recently, the composition of the gut microbiota, due to its influence on host immune system, has been linked to the effectiveness of chemotherapy and immunotherapy. Since trastuzumab, besides inhibiting the HER2 signaling, recruits innate and adaptive immune cells that mediate its cytotoxic activity in the tumor, we hypothesized that commensal bacteria can be a source of heterogeneity for the response to therapy in patients with HER2-positive breast cancer (HER2+BC). The impact of the gut microbiota on anti-HER2 therapy was studied in mice with the intestinal flora alterated by the treatment with vancomycin or streptomycin-two broad spectrum antibiotics poorly absorbed in the intestine. The association between commensal bacteria composition and clinical efficacy of trastuzumab was investigated in a cohort of HER2+BC patients treated with neoadjuvant trastuzumab. Administration of antibiotics impaired the efficacy of anti-HER2 monoclonal antibodies both in FVB and BALB/c mice bearing syngeneic mammary carcinomas expressing HER2. 16S rRNA gene profiling of FVB mouse feces showed that both antibiotics decreased bacterial α-diversity in the gut as evaluated by Chao1, Simpson and Shannon indices, lowering the abundance of Clostridiales bacteria. Mice transplanted with feces from antibiotic treated mice did not benefit from the anti-HER2 treatment supporting a direct relation between intestinal bacteria and therapeutic efficacy. Analysis by flow cytometry and immunohistochemistry of tumors grown in mice showed that alteration of gut microbiota compromised the recruitment of CD4+ T cells and Natural Killer (CD49b+, GZMB+) cells upon anti-HER2 administration. Fecal 16S rRNA gene sequencing demonstrated a significantly higher microbial α-diversity in patients who achieved a pathological complete response compared to non-responders using several indices. Moreover, a clustering effect by patient’s response was observed visualizing the β-diversity. OTUs belonging to the Clostridiales and Bacteroidales orders were reduced and enriched, respectively, in non-responders. Our data support that the composition of the gut microbiota, especially as regards the abundance of Clostridiales bacteria, has a role in the therapeutic efficacy of trastuzumab both in mice and patients. Therefore, the manipulation of intestinal bacteria may represent a new strategy to improve the cure of HER2+BC patients. (Supported by Associazione Italiana per la Ricerca sul Cancro). Citation Format: Martina Di Modica, Viola Regondi, Giorgio Gargari, Arianna Bonizzi, Stefania Arioli, Beatrice Belmonte, Claudio Tripodo, Simone Guglielmetti, Fabio Corsi, Tiziana Triulzi, Elda Tagliabue. The gut microbiota contributes to the effectiveness of HER2-targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4959.

The incidence of breast cancer among Chinese women has been historically much lower than that of Caucasian women but has increased dramatically over the past 30 years in urban areas of China, an increase likely to continue in the next decades across China with progressive urbanization and economic development. However, it is still unclear whether differences in breast cancer incidence between Chinese and Caucasian women reflect differences in disease biology in addition to environmental factors. To address this question, we compared consecutive cohorts of Chinese and Italian breast cancer patients who underwent surgical resection in the years 2005-2007 at Fudan Cancer Center of Shanghai (China) and Fondazione IRCCS Istituto Tumori of Milan (Italy), respectively, two comprehensive cancer centers with similar inpatients and outpatients recruited in a large area. Analyses of clinico-pathological characteristics of 1563 Chinese and 1052 Italian consecutive breast cancer patients revealed that 50% of patients in the Chinese cohort were younger than 50 years versus 29% of Italian patients, indicating that Chinese women develop breast cancer earlier than Italian patients, with a peak of frequency at 50-60 years. Among Chinese breast tumors, 63% were estrogen receptor (ER)-positive versus 85% of the Italians, and the prevalence of ER-positive tumors was equivalent in pre- and postmenopausal Chinese cases, suggesting the same hormonal features in tumors of younger and older patients, consistent with findings in the Italian cohort. Microarray-based gene and miRNA expression profiling was then conducted on 80 Chinese and 97 Italian samples using Illumina Gene and MicroRNA expression technologies. To minimize potential bias and artifact due to variability in specimen collection, storage, processing and analysis of high-throughput results, samples initially collected in the Chinese and Italian hospitals were all stored, randomly processed and analyzed in the Italian center. Analysis revealed a comparable pattern of gene and miRNA expression in the 2 cohorts with unsupervised hierarchical clustering of 12,338 genes and 858 miRNAs, and similar expression of intrinsic and extracellular matrix (ECM) genes led to a similar partition in intrinsic and ECM subtypes, indicating molecular similarity among Chinese and Caucasian breast cancers. Supported by Ministero Affari Esteri (Italy-China significant bilateral project). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-07-08.

Criteria for the decision to treat small tumors in adjuvant setting remain debatable, especially for HER2-positive tumors, since HER2 itself is associated with poor prognosis. Analysis of a large database including 503 T1 HER2-positive breast carcinomas treated with chemotherapy plus trastuzumab in Italian oncology clinics from 2006 to 2009 and, with 32 months of median follow-up and with 35 (7.0%) relapses, revealed no statistically significant differences in relapse rate among the T1a/T1micro, T1b and T1c groups, suggesting that tumor size is not associated with relapse probability in T1 HER2-positive tumors; the only clinico-pathological factors significantly associated with disease-free survival were nodal involvement (unadjusted HR = 3.4, 95%CI = 1.6-7.3, p = 0.002), loss of hormone receptor expression (HR = 3.1, 95%CI = 1.5-6.2, p = 0.002) and high tumor grade (HR = 2.9, 95%CI = 1.2-6.9, p = 0.020). Multivariate analysis indicated that patients who were both ER-negative and lymph node-positive presented the highest risk of relapse (HR = 5.5, 95%CI = 2.8-10.9, p = 1 cm, our data identify T1 tumor ER-negativity/axillary lymph node-positivity as the crucial criteria for adjuvant treatment, independent of tumor size categories. Supported by AIRC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-06.

Trastuzumab, a recombinant humanized monoclonal antibody directed to the HER2 protein, has shown survival benefits in women with HER2-positive breast cancer, and treatment is now FDA-approved in combination with chemotherapy. Nonetheless, some patients relapse after treatment, underscoring the need to identify patients for whom chemotherapy + trastuzumab is adequate versus patients requiring additional drugs. To search for factors predictive of relapse in HER2-positive breast carcinoma patients treated adjuvantly with trastuzumab, we conducted gene expression profiling analysis in 53 cases selected among a cohort of 243 patients (32 of whom relapsed) treated in the clinic with chemotherapy + trastuzumab in our institute during 2006-2009, with median follow-up of 32 months; the 53 cases comprised 23 of the relapsed and 30 of the non-relapsed patients with similar clinico-pathological characteristics (size, lymph node involvement and estrogen receptor positivity) for whom sufficient material was available. RNA extracted from formalin-fixed, paraffin-embedded (FFPE) was profiled using HumanHT12_v4 wgDASL expression BeadChips (Illumina). A Cox's proportional hazard model was used to estimate the association between gene expression and relapse-free survival (RFS), and a multivariate permutation test was used to check the proportion of false discoveries. Analysis identified 330 probes corresponding to 308 unique genes as significantly associated to RFS (a We also tested the feasibility of developing a predictive model including the gene expression data. Based on 10-fold cross-validation, our model was able to stratify patients into two groups (high and low risk), with a 4-year RFS probability of 14.5% in high-risk versus 87% in low-risk tumors (HR = 8.33, 95% CI = 3.53-18.18, p Together, our results suggest the usefulness of molecular characteristics of primary tumors as indicators of early relapse in patients treated adjuvantly with trastuzumab and as tools to identify patients for whom additional treatments are required. Supported by AIRC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-03.

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.

Trastuzumab, a recombinant humanized monoclonal antibody directed to the HER2 protein, has shown survival benefits in women with HER2-positive breast cancer, and treatment is now FDA-approved in combination with chemotherapy. However, some patients do not respond clinically to trastuzumab, pointing to the need for further definition of trastuzumab killing activity on tumor cells to optimize this therapy. Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in HER2-positive breast cancer patients, but the mechanism(s) underlying this synergy remains unclear. Because drug-stressed cells can dynamically regulate factors that favor activity of immune effector cells, and because trastuzumab-mediated ADCC is crucially dependent on NK cell activity, we hypothesize that this synergy reflects enhanced trastuzumab-mediated ADCC on tumor cells due to improved rather than impaired participation of immune effectors resulting from drug-induced stress. To test this hypothesis, we investigated the effect of chemotherapy in HER2-positive breast carcinoma models on NK receptor ligands and correlated the changes in these molecules with the ability of trastuzumab to mediate ADCC. Flow cytometry revealed a 4-fold upmodulated surface expression of NKG2D (MICA, MICB, ULBP1, ULBP2) and DNAM-1 (CD112 and CD155) ligands in HER2-positive breast carcinoma cell lines BT474 and MDA-MB-361 treated for 6 hr with taxotere at a 72-hr IC50 dose, consistent with results of Western blot analysis using soluble lysates obtained from chemotherapy-treated HER2-positive breast carcinoma xenografts. The enhanced expression of NKG2D and DNAM-1 ligands in breast carcinoma cells was accompanied by about a 50% increase in trastuzumab-mediated in vitro ADCC in chemotherapy-treated versus untreated cells. Antibodies blocking NKG2D and DNAM-1, but not control monoclonal antibody, abolished the chemotherapy-induced increase of in vitro trastuzumab-dependent ADCC. Increased expression of NK ligands in taxotere-treated BT474 cells was associated with cytoskeletal damage assessed by confocal microscopy; at 72 hr after chemotherapy, NK ligands returned to basal levels, pointing to the importance of duration of NK ligand enhancement for the chemotherapy-induced trastuzumab-mediated ADCC. Together, our results indicate that chemotherapy can modulate expression levels of NK-activating ligands on human breast carcinoma cells in correlation with trastuzumab-mediated ADCC, raising the possibility of identifying optimal chemotherapy administration schedules to maximize activity of trastuzumab-mediated ADCC effectors. (Partially supported by AIRC) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-18-10.

Background. We recently demonstrated that primary breast tumors can be classified based upon extracellular matrix composition (ECM) (Bergamaschi et al.) suggesting stroma characteristics could influence tumor progression. Aim of this study is to further investigate the robustness of this classification and its impact in tumor progression and response to therapy. Methods. The expression profile of ECM-related genes was analyzed by unsupervised hierarchical clustering in 10 independent datasets of breast tumors, counting more than 1000 samples and the prognostic and predictive value of this signature were evaluated in two datasets of not treated patients and in one dataset of neo-adjuvant treated patients, respectively. Results. Only one of ECM subsets (ECM3) showed a homogeneous gene pattern that consistently allowed the classification of an independent group of tumors in all tested datasets. ECM3 is characterized by highly correlated over expression of 34 ECM genes encoding mainly structural proteins. From 24 to 38% of cases were ECM3-enriched and were mainly estrogen receptor-positive and low grade (p Moreover analysis of a data set of patients treated neoadjuvantly with chemotherapy revealed that, among the grade III tumors, pathological complete response was reached by 9% of ECM3 versus 74% of non-ECM3 tumors (p=0.04). In differentiated tumors no response difference was observed according to ECM. Conclusions. Our results provide evidence that breast carcinoma progression and response to therapy are influenced by the interaction between tumor and stromal characteristics. (Partially supported by AIRC). Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-14-06.

Synthetic oligodeoxynucleotides expressing CpG motifs (CpG-ODN) are a Toll-like receptor 9 (TLR9) agonist that can enhance the antitumor activity of DNA-damaging chemotherapy and radiation therapy in preclinical mouse models. We hypothesized that the success of these combinations is related to the ability of CpG-ODN to modulate genes involved in DNA repair. We conducted an in silico analysis of genes implicated in DNA repair in data sets obtained from murine colon carcinoma cells in mice injected intratumorally with CpG-ODN and from splenocytes in mice treated intraperitoneally with CpG-ODN. CpG-ODN treatment caused downregulation of DNA repair genes in tumors. Microarray analyses of human IGROV-1 ovarian carcinoma xenografts in mice treated intraperitoneally with CpG-ODN confirmed in silico findings. When combined with the DNA-damaging drug cisplatin, CpG-ODN significantly increased the life span of mice compared with individual treatments. In contrast, CpG-ODN led to an upregulation of genes involved in DNA repair in immune cells. Cisplatin-treated patients with ovarian carcinoma as well as anthracycline-treated patients with breast cancer who are classified as “CpG-like” for the level of expression of CpG-ODN modulated DNA repair genes have a better outcome than patients classified as “CpG-untreated-like,” indicating the relevance of these genes in the tumor cell response to DNA-damaging drugs. Taken together, the findings provide evidence that the tumor microenvironment can sensitize cancer cells to DNA-damaging chemotherapy, thereby expanding the benefits of CpG-ODN therapy beyond induction of a strong immune response. Cancer Res; 71(20); 6382–90. ©2011 AACR.

An increasing amount of experimental evidence shows 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. HER2 overexpression is a hallmark of a particularly aggressive subset of breast tumors, and its activation is strictly dependent on the trans-interaction with other members of HER family; in particular, the activation of the PI3K/Akt survival pathway, so critically important in tumorigenesis, is predominantly driven through phosphorylation of the kinase-inactive member HER3. Here, we show that miR-205, down-modulated in breast tumors compared with normal breast tissue, directly targets HER3 receptor, and inhibits the activation of the downstream mediator Akt. The reintroduction of miR-205 in SKBr3 cells inhibits their clonogenic potential and increases the responsiveness to tyrosine-kinase inhibitors Gefitinib and Lapatinib, abrogating the HER3-mediated resistance and restoring a potent proapoptotic activity. Our data describe miR-205 as a new oncosuppressor gene in breast cancer, able to interfere with the proliferative pathway mediated by HER receptor family. Our study also provides experimental evidence suggesting that miR-205 can improve the responsiveness to specific anticancer therapies. [Cancer Res 2009;69(6):2195–200]

Preliminary results from a pilot trial on trastuzumab's mechanism of action against operable breast tumors overexpressing Her2 suggested a role for antibody-dependent cell cytotoxicity (ADCC). To examine factors affecting ADCC intensity and variability, we extended this study to the phenotypic and functional analysis of circulating mononuclear cells in 18 patients. ADCC was induced by trastuzumab therapy in 15 of 18 patients (83%). Inability to develop ADCC in three patients did not depend on inadequate levels of trastuzumab because further increase in its concentration in vitro was ineffective. Rather, susceptibility to develop ADCC was fairly predicted by test with trastuzumab before therapy and was correlated to the number of lymphocytes coexpressing CD16 and CD56. Phenotypic analysis at the end of ADCC evaluating down-regulation of CD16, and up-regulation of CD69 and CD107a, confirmed that natural killer (NK) cells and CD56+ T cells were involved in productive engagement of trastuzumab. Also, the killing efficiency of CD16+ lymphocytes was influenced by 158 V/F polymorphism of FcγRIII (CD16), whereas variations of CD247 on NK cells were consistent with trends between ADCC before and after therapy. Complete pathologic response was observed in one patient showing ADCC of outstanding intensity, whereas four cases of partial response showed intermediate ADCC; none of the three patients unable to mount ADCC had significant tumor regression. These data indicate that quantity and lytic efficiency of CD16+ lymphocytes are major factors for ADCC induction by trastuzumab, and confirm that breast cancer responses to short-term trastuzumab monotherapy may depend on involvement of the ADCC mechanism. [Cancer Res 2007;67(24):11991–9]

In some HER2-positive breast tumors, cell surface overexpression of HER2 is not associated with gene amplification but may instead rest in altered gene transcription, half-life, or recycling of the oncoprotein. Here, we show that HER2 overexpression in HER2 2+ carcinomas is associated with neither an increase in gene transcription nor a deregulation in the ubiquitin-dependent pathways, but instead seems to be regulated by protein kinase Cα (PKCα) activity. The stimulation of PKCα up-regulated HER2 expression, whereas PKCα inhibition by pharmacologic treatments and PKCα-specific small interfering RNA led to a dramatic down-regulation of HER2 levels only in breast cancer cells HER2 2+. Consistent with the in vitro data, our biochemical analysis of HER2 2+ human primary breast specimens revealed significantly higher levels of phosphorylated PKCα compared with HER2-negative tumors. Inhibition of HER2 activation by the tyrosine kinase inhibitor lapatinib led to decreased levels of PKCα phosphorylation, clearly indicating a cross-talk between PKCα and HER2 molecules. These data suggest that HER2 overexpression in HER2 2+ carcinomas is due to an accumulation of the recycled oncoprotein to the cell surface induced by activated PKCα. [Cancer Res 2007;67(11):5308–17]

INTRODUCTION: Taking advantage of aerosol for repeated local applications in the lung, we previously demonstrated that combination of Poly(I:C) and CpG-ODN, TLR3 and 9 ligands capable to activate innate effectors, induced a significant antitumor effect against B16 melanoma lung metastases, which correlated with a strong activation of NK cells. We investigated the sequence of events that lead to NK cells activation by aerosolized TLR agonists in the lung. Although NK cells express certain TLRs, controversies exist regarding their direct responsiveness or dependence on macrophages. Alveolar macrophages have several unique features both promoting host defense mechanisms against invading microorganisms, but also establishing an immunosuppressive microenvironment to avoid inflammatory responses to inhaled particles. Our findings, establishing the sequence of events of lung immune cells activation, might represent the basis for the development of combined aerosol therapies that favor NK cell triggering. EXPERIMENTAL PROCEDURE: Alveolar macrophages and NK cells were recovered by bronchoalveolar lavage and spleen of C57BL/6 mice, respectively. Mice i.v. injected with 5×105 B16 cells were treated starting 7 days later every three days with aerosolized Poly(I:C)/CpG-ODN alone or combined to nebulized myeloid derived suppressive cells (MDSC)-depleting RB6-8C5 antibody. Immune cell populations and expression of M1/M2 markers were analysed by flow cytometry and Real Time PCR, respectively, in digested lungs. Cytotoxic activity of NK cells was assessed by 51Cr release assay. RESULTS: In vitro TLR3/TLR9 stimulation induced IFN-γ secretion by naïve NK cells, but an increase in their cytotoxicity was detected only when NK cells were cocultured with alveolar macrophages pretreated with the two agonists. Alveolar macrophages from melanoma lung metastases-bearing mice treated with aerosolized TLR agonists also promoted NK cell cytotoxicity ex vivo. Moreover, naïve alveolar macrophages incubated with activated NK cells from lungs of melanoma metastases-bearing mice aerosolized with TLR9/TLR3 agonists, up-regulated M1- and downregulated M2-related genes, suggesting a bidirectional crosstalk between NK cells and macrophage. Thus, TLR agonists-activated lung macrophages promote NK cell cytotoxicity and, reciprocally, NK cells have the potential to shape the macrophage programming to M1-like phenotype. Our results also indicated that this positive interplay can be further improved by the depletion of myeloid derived suppressive cells that exert a negative effect on macrophage activation. CONCLUSION: This study points to the promise of combinations of immunotherapeutic agents delivered locally by inhalation to promote macrophages-NK cell activation in the lung as a novel strategy to treat lung cancer patients. Citation Format: Michele Sommariva, Valentino Le Noci, Elda Tagliabue, Andrea Balsari, Lucia Sfondrini. Activation of NK cells cytotoxicity mediated by alveolar macrophages in the lung against murine melanoma metastases by combined aerosol immunotherapy [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 3699. doi:10.1158/1538-7445.AM2017-3699

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.

Interactions between cancer cells and the surrounding microenvironment are crucial determinants of cancer progression. Fibroblasts isolated from carcinomas (cancer associated fibroblasts, CAF) have been shown to fuel the neoplastic process. While normal fibroblasts from different organs generally present very specific transcriptional programs, CAF from different tumors display a similar phenotype reminiscent of myofibroblasts found at sites of wound healing, indicating that common transcriptional programs could be induced by cancer cells on the surrounding microenvironment. To investigate the existence of common mechanisms in fibroblast activation across different cancer types, gene expression profiles of tumor stroma or cancer associated fibroblasts (CAF) derived from breast, prostate and lung cancer were previously evaluated performing an inter-pathology bio-informatics analysis. For each comparison, genes were ranked and subjected to Gene Set Enrichment Analysis to identify Canonical Pathways significantly enriched. Most of commonly enriched gene sets were related to extracellular matrix (ECM) structure and remodeling, cytoskeletal organization and cell-cell adhesion. We specifically evaluated a previously identified ECM-related signature (ECM3) defining a breast cancer subtype likely to progress. We found that ECM3 signature was significantly enriched (p-value < 0.05) in cancer associated fibroblasts compared to the normal counterpart across different pathologies supporting the implication of ECM-related genes in fibroblasts with activated status. Furthermore increased expression of some of the most ECM3 genes (COL10A1, COMP and COL11A1) was detected in fibroblasts co-cultured with breast cancer cells, both in cellular extracts and in the conditioned medium (CM), suggesting their potential importance as circulating markers for early detection and risk assessment. To explore the significance of ECM-related molecules also in lung, we finally selected some of enriched ECM3-related molecules in lung fibroblasts (SPARC, COL11A1, MMP11, FN1, COMP, COL10A1) and we evaluated their presence in the conditioned medium. The analysis showed that the ECM molecules could be detected in medium conditioned by lung fibroblasts and generally enriched in CAF_CM compared to NF_CM (SPARC p = 0.004, COMP p = 0.003, MMP11 p = 0.012). Interestingly, analysis of de-cellularized extracellular matrix showed that Fibronectin 1 (FN1) was preferentially deposited by CAFs compared to NFs (p = 0.003) denoting a possible role of this molecule in the modulation of extracellular matrix during tumor development and/or progression. These findings support the hypothesis that identification of common factors responsible for proficient tumor-stroma cross-talk could be instrumental in novel strategies for risk assessment in different diseases. Citation Format: Francesca Andriani, Marta Giussani, Federica Facchinetti, Maurizio Callari, Elda Tagliabue, Ugo Pastorino, Luca Roz, Gabriella Sozzi, Gabriella Sozzi. Cancer-associated fibroblasts transcriptional pattern reveals common changes in extracellular matrix-related genes across different cancer types. [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 5085.

It is now well-established that the local microenvironment of an emerging tumor plays a vital role in various steps of tumorigenesis. Stromal cells, such as fibroblasts, endothelial and various inflammatory cells, can contribute to tumor progression, and tumor cells can directly model their microenvironment, influencing host cells to secrete factors that favor cancer cell survival and growth. Recent studies have demonstrated the existence of cross-talk between breast cancer (BC) cells and adipocytes, the most abundant stromal cell in the breast, supporting their involvement in BC and consistent with epidemiological studies reporting the association of obesity and insulin resistance/diabetes with a higher risk and poor prognosis of BC. Based on this evidence, we investigated whether adipocyte de-differentiation is the crucial trigger in inducing pro-tumor microenvironment remodeling and tumor progression. Co-culture of 4T1 mouse mammary tumor cells using Transwell or their conditioned medium with mature adipocytes derived from the 3T3-L1 mouse cells induced adipocyte de-differentiation, observed as a reduced expression of specific differentiation markers (peripilin, adipocyte protein 2 (aP2), PPARã and adiponectin) in Western blots and by qRT-PCR, and a reduced triglyceride content as assessed by oil red O staining compared to mature adipocytes. Strong adipocyte de-differentiation, associated with an increase in pro-inflammatory molecules (IL6 and CCL2), was also induced in vitro by tumor interstitial fluids obtained from MMT-PyMT transgenic mice, but not by plasma of these mice. Preliminary studies to identify molecules involved in the cross-talk indicated that the phenomenon is exosome-unrelated. Based on the relevant role of the PPARã transcription factor in inducing and maintaining adipocyte differentiation, we co-cultured adipocytes with 4T1 BC cells in the presence of several PPARã agonists enriched in the diets of many populations displaying a low incidence of cancer (þ3-fatty acids), and in the presence of glitazones (rosiglitazone and pioglitazone) to force adipocyte stable differentiation. While most of these compounds did not block adipocyte de-differentiation induced by tumor cells, pioglitazone and eicosanoid acid showed some activity in inhibiting this de-differentiation in vitro. Treatment of MMTV-PyMT transgenic mice at tumor onset by oral gavage with pioglitazone did not significantly reduce mammary tumor growth compared to controls, whereas eicosanoid acid treatment significantly reduced tumor multiplicity and tumor burden compared to controls. Overall, our results suggest that tumor-induced adipocyte de-differentiation participates in tumorigenesis and that the blockade of this program can inhibit tumor progression. Supported by AIRC and Fondazione Cariplo. Citation Format: Tiziana Triulzi, Valentina Ciravolo, Viola Regondi, Martina Di Modica, Claudia Chiodoni, Elda Tagliabue. Targeting the crosstalk between tumor cells and adipocytes to block breast cancer progression. [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 748.

The transcription factor Autoimmune Regulator (AIRE) gene plays a fundamental role in tolerance process by promoting the ectopic expression of thousands of genes encoding for tissue-specific antigens (TSAs) in medullary thymic epithelial cells (mTECs). Beside the high expression of AIRE in mTECs, there are few evidences of AIRE expression in other lymphoid organs and very recently, AIRE was described to be expressed in human and mouse keratinocytes and in tumors originating in stratified and pseudostratified epithelia. Therefore, we evaluated the hypothesis that AIRE could be expressed by epithelial tumors and we focused on breast cancer, for which large public datasets are available. Our analysis on the TCGA, the largest public breast cancer RNA-seq dataset, revealed that AIRE is expressed in 33% of the cases, and Aire protein expression was confirmed in 26% of 115 human primary breast cancer specimens by immunohistochemistry analysis. AIRE expression was revealed associated to a better relapse-free survival in the TCGA patients and also in two public gene expression microarray-based dataset developed on Agilent and Illumina platform (NKI-295 and KM-plotter). In all the datasets analyzed, AIRE expression resulted an independent strong prognostic factor for relapse-free survival, particularly in ER-positive tumors. Ingenuity Pathway Analysis (IPA) showed that AIRE-expressing tumors were enriched in translation-related pathways, accordingly with AIRE's described function in mTECs. Moreover, in breast cancer luminal cell lines MCF-7 and MDA-MB-361 transfected with AIRE-expressing vector, a significant increase of cell in G1 phase and activation of caspases cascade were observed, supporting the hypothesis that genotoxic stress, induced by AIRE-mediated proteins over-translation, leads to cycle arrest and apoptosis. These data highlight for the first time AIRE expression in breast cancer and an association with a better prognosis was revealed. Imbalance of cellular homeostasis, caused by AIRE transcriptional function, is a new unusual mechanisms to trigger apoptosis in breast cancer cells. Citation Format: Francesca Bianchi, Loris De Cecco, Tiziana Triulzi, Sandra Romero-Cordoba, Michele Sommariva, Chiara Storti, Piera Aiello, Elda Tagliabue, Andrea Balsari, Lucia Sfondrini. AIRE is expressed and associated with good prognosis in 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 3924.

Background: Almost 90% of all breast cancers (BCs) express a splice variant of HER2 lacking exon 16 (d16HER2), which promotes the generation of stable and active d16HER2 homodimers on the tumor cell surface. Comparison of the tumorigenic potential of human d16HER2 and the full-length HER2 (WTHER2) in transgenic mice revealed a significantly shorter latency period and a higher incidence of tumors in the d16HER2 line, suggesting enrichment of this variant in HER2+ BC stem cells. Based on reports that cruciferous vegetable-derived compounds such as isothiocyanates (ITCs) mediate strong anti-tumorigenic effects on numerous oncotypes and target BC stem cells, we tested the effect of the phenethyl ITC (PEITC), alone or together with Trastuzumab (T), which reportedly targets HER2+ BC stem cells, on d16HER2-positive tumor growth and progression in transgenic mice. Methods: The percentage of tumor cells expressing high levels of CD29 and CD24 and low levels of SCA-1 (CD29high/CD24+/SCA-1low) murine stem cell markers was evaluated by multiparametric flow cytometry in cell models derived from spontaneous d16HER2 and WTHER2 transgenic lesions. Mammosphere forming efficiency (MFE%) was calculated in d16HER2- and WTHER2-positive tumor cells. The therapeutic activity of PEITC, T and their combination was assessed in vitro based on their ability to decrease the MFE% and the expression of stem cell markers in d16HER2- vs. WTHER2-positive tumor cells, and in vivo in d16HER2 transgenic mice. Analyses by western blot, histology/immunohistochemistry and magnetic resonance spectroscopy (MRS) are ongoing to assess apoptosis, necrosis and metabolomic profiles of treated tumors vs. controls. Results: d16HER2 tumor cells showed a significantly higher MFE% and higher expression of stem cell markers than did WTHER2 tumor cells. PEITC and T significantly reduced MFE% as compared to controls (p = 0.0342 and p = 0.0083, respectively), and their combination exerted significantly stronger inhibition than T (p = 0.001) or PEITC (p = 0.0003) alone only in d16HER2 cells. The percentage of tumor cells expressing CD29high/CD24+/SCA-1low in d16HER2 tumor cells after treatment with T, PEITC or both was also reduced. Whereas PEITC monotherapy did not significantly reduce mammary tumor incidence compared to controls, while T effectively suppressed d16HER2-driven tumor growth (p = 0.0002), the combination of PEITC and T significantly heightened the impairment of mammary lesion development compared to T alone (p = 0.0046). Ex vivo metabolomic analyses by MRS of d16HER2-positive murine and human BC cell lines showed an increase in glycolytic pathway as compared to the respective WTHER2 cells. Conclusions: Our results provide in vivo evidence that the combination of T and PEITC mediates a significantly greater anti-tumor effect than either alone, most likely targeting BC stem cells. Supported by the Italian Ministry of Health and AIRC. Citation Format: Ada Koschorke, Lorenzo Castagnoli, Gaia C. Ghedini, Tiziana Triulzi, Claudia Chiodoni, Rossella Canese, Egidio Iorio, Manuela Iezzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. Phenethyl isothiocyanate hampers growth and progression of HER2-positive breast cancers. [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 3826.

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.

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.

Doxorubicin treatment was found to augment the expression of the extracellular matrix (ECM) protein fibulin-1 in cultured human breast cancer cell lines and in MDA-MB-361 tumors grown in athymic mice. Doxorubicin was also found to augment tumor expression of the fibulin-1–binding proteins fibronectin and laminin-1. Growth of breast cancer cell lines on Matrigel, an ECM extract containing fibulin-1 and laminin-1, resulted in lower levels of doxorubicin-induced apoptosis as compared with controls. Moreover, tumors formed by injection of athymic mice with MDA-MB-361 cells mixed with Matrigel were significantly more doxorubicin resistant and displayed lower levels of apoptosis compared with those that formed in the absence of Matrigel. Monoclonal antibodies against fibulin-1 reversed Matrigel-dependent doxorubicin resistance. Furthermore, small interfering RNA–mediated suppression of fibulin-1 expression in breast cancer cells resulted in a 10-fold increase in doxorubicin sensitivity as compared with control cells. Together, these findings point to a role for fibulin-1 in breast cancer chemoresistance. [Cancer Res 2007;67(9):4271–7]

The transmembrane tyrosine kinase receptor HER2, overexpressed in ∼20% of human breast cancers (BCs), identifies an aggressive tumor subtype and is reportedly an important regulator of breast cancer-initiating cell activity (BCIC). We found that ∼90% of HER2+ BC patients constitutively express a splice isoform of wild-type HER2 (WTHER2) gene characterized by the lack of exon 16 (d16HER2), a deletion that promotes the generation of a particularly aggressive HER2 isoform and that forms stable and constitutively activated d16HER2 homodimers. Our comparison of the tumorigenic potential of the human d16HER2 and WTHER2 genes in the corresponding transgenic mouse models revealed a significantly shorter tumor latency period (p< 0.001) and a higher tumor incidence in the d16HER2 mice (p Supported by Minister of Health and AIRC Citation Format: Lorenzo Castagnoli, Ada Koschorke, Gaia C. Ghedini, Lorenzo Galvani, Valentina Ciravolo, Cristina Ghirelli, Arianna Palladini, Alessia Lamolinara, Manuela Iezzi, Pier Luigi Lollini, Tiziana Triulzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. d16HER2 splice variant regulates the activity of HER2-positive breast cancer-initiating cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2015-2314