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Supplementary Information includes Supplementary Materials and Methods, Supplementary Figures 1-7, with Legends and Supplementary References. Supplementary Figure S1: Loss of stathmin impairs mouse mammary gland development. Supplementary Figure S2: Loss of stathmin decreases the growth of mammary epithelial cells. Supplementary Figure S3: Loss of stathmin alters the mammary gland organization. Supplementary Figure S4: mRNA levels of STAT5 target genes are differently modulated when stathmin is silenced. Supplementary Figure S5: Stathmin expression correlates with poor prognosis in breast cancer patients. Supplementary Figure S6: Î"16HER2-driven mammary neoplastic lesions display luminal features. Supplementary Figure S7: Absence of stathmin in pre-neoplastic mouse mammary glands (13 weeks of age) weakens the activation of Î"16HER2 transformation-related pathways

Supplementary Table S1: Molecular and Pathological variables of patients included in the discovery cohort.

Table S1: Clinical Data of BC samples collected at Centro di Riferimento Oncologico di Aviano (CRO), IRCCS, National Cancer Institute, Aviano, Italy. Table S2: Clinical Data of BC samples collected in University "Sapienza" of Rome, Santo Andrea Hospital, Rome, Italy

NMuMG CTR cells

Postnatal development of the mammary gland relies on the maintenance of oriented cell division and apicobasal polarity, both of which are often deregulated in cancer. The microtubule (MT) network contributes to control these processes; however, very little is known about the impact of altered MT dynamics in the development of a complex organ and on the role played by MT-interacting proteins such as stathmin. In this study, we report that female stathmin knock-out (STM KO) mice are unable to nurse their litters due to frank impairment of mammary gland development. In mouse mammary epithelial cells, loss of stathmin compromised the trafficking of polarized proteins and the achievement of proper apicobasal polarity. In particular, prolactin receptor internalization and localization was altered in STM KO mammary epithelial cells, leading to decreased protein stability and downmodulation of the Prl/PrlR/STAT5 signaling pathway. Absence of stathmin induced alterations in mitotic spindle orientation, accumulation of mitotic defects, and apoptosis, overall contributing to tissue disorganization and further decreasing the expansion of the mammary epithelial compartment. Loss of stathmin in MMTV-Δ16HER2 transgenic mice decreased the incidence and increased the latency of these very aggressive mammary carcinomas. Collectively, these data identify the essential mammary protein stathmin as protumorigenic and suggest it may serve as a potential therapeutic target in breast cancer.Significance:Stathmin expression is critical to maintain oriented cell division and apicobasal polarity in normal mammary glands and to establish a protumorigenic program that eventually sustains HER2-positive breast cancer formation in mice.

Supplementary Figure S1. Validation of differentially expressed miRs in the discovery dataset. Supplementary Figure S2. Design of a recurrence prediction model in HNSCC based on miRs expression using a bioinformatic approach. Supplementary Figure S3. In vitro validation of miR-9 putative targets Supplementary Figure S4. Validation of putative miRs seed sites in the 3'-UTR of SASH1 and SP1 genes Supplementary Figure S5. The combined expression of miRs-1, 133a and -150 is necessary to downregulate SP1 mRNA and protein levels. Supplementary Figure S6. Correlation analyses of miRs putative targets in primary HNSCC collected in our Institute and described in Supplementary table S2 as validation set. Supplementary Figure S7. Prognostic significance of 4 miRs-targets in the TCGA HNSCC dataset. Supplementary Table S3. Median miRs' expression (qRT-PCR) according to selected characteristics Supplementary Experimental Procedures

miR-223 is an anti-inflammatory miRNA that in cancer acts either as an oncosuppressor or oncopromoter, in a context-dependent manner. In breast cancer, we demonstrated that it dampens the activation of the EGF pathway. However, little is known on the role of miR-223 during breast cancer onset and progression. miR-223 expression was decreased in breast cancer of luminal and HER2 subtypes and inversely correlated with patients' prognosis. In normal luminal mammary epithelial cells, miR-223 acted cell autonomously in the control of their growth and morphology in three-dimensional context. In the MMTV-Δ16HER2 transgenic mouse model, oncogene transformation resulted in a timely abrogation of miR-223 expression, likely due to activation of E2F1, a known repressor of miR-223 transcription. Accordingly, treatment with CDK4/6 inhibitors, which eventually results in restraining E2F1 activity, restored miR-223 expression and miR-223 ablation induced luminal breast cancer resistance to CDK4/6 inhibition, both in vitro and in vivo. Notably, miR-223 expression was lost in microdissected ductal carcinoma in situ (DCIS) from patients with luminal and HER2-positive breast cancer. Altogether, these results identify downmodulation of miR-223 as an early step in luminal breast cancer onset and suggest that it could be used to identify aggressive DCIS and predict the response to targeted therapy.Significance:miR-223 may represent a predictive biomarker of response to CDK4/6 inhibitors and its loss could identify DCIS lesions that are likely to progress into invasive breast cancer.

Supplementary Information, Methods and Figures. Supplementary Figure 1. Loss of miR-223 does not alter the murine mammary gland architecture; Supplementary Figure 2. miR-223 KO mice do not display any proliferative advantage during â^†16HER2-driven mammary tumorigenesis; Supplementary Figure 3. miR-223 WT and KO â^†16HER2 tumor-derived mammary epithelial cells (mMECs) display comparable tumorigenic potential; Supplementary Figure 4. miR-223 partially counteracts HER2-driven BC cell proliferation and 3D-organization; Supplementary Figure 5. miR-223 is rapidly down-modulated during cell transformation; Supplementary Figure 6. miR-223 is upregulated in response to anti-proliferative stimuli

Purpose: Head and neck squamous cell carcinomas (HNSCCs) cause more than 300,000 deaths worldwide each year. Locoregional and distant recurrences represent worse prognostic events and accepted surrogate markers of patients' overall survival. No valid biomarker and salvage therapy exist to identify and treat patients at high-risk of recurrence. We aimed to verify if selected miRNAs could be used as biomarkers of recurrence in HNSCC.Experimental Design: A NanoString array was used to identify miRNAs associated with locoregional recurrence in 44 patients with HNSCC. Bioinformatic approaches validated the signature and identified potential miRNA targets. Validation experiments were performed using an independent cohort of primary HNSCC samples and a panel of HNSCC cell lines. In vivo experiments validated the in vitro results.Results: Our data identified a four-miRNA signature that classified HNSCC patients at high- or low-risk of recurrence. These miRNAs collectively impinge on the epithelial–mesenchymal transition process. In silico and wet lab approaches showed that miR-9, expressed at high levels in recurrent HNSCC, targets SASH1 and KRT13, whereas miR-1, miR-133, and miR-150, expressed at low levels in recurrent HNSCC, collectively target SP1 and TGFβ pathways. A six-gene signature comprising these targets identified patients at high risk of recurrences, as well. Combined pharmacological inhibition of SP1 and TGFβ pathways induced HNSCC cell death and, when timely administered, prevented recurrence formation in a preclinical model of HNSCC recurrence.Conclusions: By integrating different experimental approaches and competences, we identified critical mediators of recurrence formation in HNSCC that may merit to be considered for future clinical development. Clin Cancer Res; 23(14); 3769–80. ©2017 AACR.

The lack of suitable experimental approaches to investigate and model functional heterogeneity in vivo has had a profound negative impact on our understanding of how heterogeneity affects the response to immunotherapy. To bridge this technological gap, we leverage a new platform that visualizes the spatial architecture of subclones and microenvironments. To study heterogeneous populations of cells and their clonal dynamics in vivo, we performed barcode lineage tracing with next-generation sequencing analysis. It allows a quantitative evaluation of the spatial distribution and temporal clonal dynamic in vivo with multiple pharmacological perturbations, such as immune checkpoint blockade (ICB). We found that treatment with a PD1 inhibitor, despite a limited effect on tumor volume, induced dramatic changes in tumor clonal architecture. Furthermore, we revealed that subclones from well-defined geographical domains in vivo share differential behavior upon immune checkpoint blockade (ICB). By visualizing 40 different markers with multiplexed staining technology, we further identified that the microenvironment also exhibits a high level of spatial heterogeneity. Therefore, we coupled spatial barcode sequencing with high-content imaging and revealed the differential microenvironment feature of sensitive and resistant clones. We will further explore the mechanisms of immune evasion at the subclonal level. Through isolation and deep characterization of clonal lineages endowed with a distinct ability to engage the immune response, we hope to identify new vulnerabilities to overcome resistance to ICB. Citation Format: Er-Yen Yen, I-Lin Ho, Chieh-Yuan Li, Charles Dyke, Shan Jiang, Francesca Citron, Sergio Attanasio, Rutvi Shah, Ko-Chien Chen, Giulio Draetta, Andrea Viale. Uncovering key microenvironmental features for immunotherapy response at the subclonal level in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5868.

Introduction: DNA replication is a tightly regulated, multi-step process involving licensing and replication factors that faithfully orchestrate a single duplication of the entire genome exclusively during S phase of the cell cycle. Although the mechanisms that prevent uncoordinated DNA replication are well characterized, it remains unclear how cells counteract its potentially harmful effects when it does occur, such as in cancer cells driven by aberrant oncogenic signaling and loss of cell cycle checkpoints. Here, we identify DPY30, an understudied component of all COMPASS complexes, as a modulator of DNA replication. We found upregulated DPY30 expression in pancreatic ductal adenocarcinoma (PDAC) as well as a correlation between increasing DPY30 expression level and higher tumor grade and poor clinical outcomes. Experimental procedure: Human- and mouse-derived PDAC cells silenced or knockout for DPY30 expression were generated and characterized for their in vivo growth in recipient mice with increasing level of immune pressure. Chromatin immunoprecipitation followed by massive parallel sequencing was used to measure the epigenetic landscape in DPY30 or WDR5 knockdown cells. Unbiased proteomics and transcriptomics analyses were performed to elucidate molecular mechanisms. Results: The canonical function of COMPASS complexes is to regulate histone methylation. Unexpectedly, DPY30 loss did not affect histone methylation patterns compared to WDR5 loss, the core member of the COMPASS-like complex. This prompted unbiased proteomics analysis, which showed that DPY30 interacts with members of the DNA helicase, possibly stabilizing the DNA replication fork progression. We found that DPY30 overexpression, by preventing Geminin ubiquitylation, strongly stabilizes its expression, which in turn suppresses Cdt1 activity in late S-phase, buffering the risk of uncoordinated DNA replication in G2/M phase. Consistently, DPY30 loss resulted in DNA re-replication and chromosomal instability, as manifested by DNA fiber and metaphasic spread. In vivo, whole-exome sequencing analysis of DPY30-knockout tumors revealed that DPY30 loss markedly increased mutational burden in immunodeficient mice. In syngeneic models, DPY30 loss strongly inhibited tumor growth, prolonged survival, and increased T cell infiltration of tumors. Moreover, the immune system selectively cleared tumor cells with complex karyotypes, and we observed improved anti-tumor efficacy with DPY30 knockdown and anti-PD-1 treatment compared to either condition alone. Conclusions: In summary, we have identified a previously undescribed function for DPY30 to stabilize the replisome machinery and prevent excessive DNA replication. Our findings indicate that, in PDAC, DPY30 promotes genome stability, thus providing a rationale for targeting DPY30 or its effector proteins in combination with immune-checkpoint inhibitors. Citation Format: Francesca Citron, Luigi Perelli, Yanshuo Chu, Sanjana Srinivasan, I-Lin Ho, Er-Yen Yen, Rutvi Shah, Sergio Attanasio, Luis Castillo Montanez, Mauro Di Pilato, Shan Jiang, Wantong Yao, Sisi Gao, Angela Deem, Giannicola Genovese, Linghua Wang, Virginia Giuliani, Andrea Viale, Giulio F. Draetta. DPY30 loss leads to DNA re-replication and immunoediting in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 303.

Sulfation of proteins, carbohydrates, lipids, and xenobiotics is an essential post-translational modification (PTM) process thought to play critical roles in diverse biological processes ranging from detoxification, cell signaling, and extracellular matrix architecture, to immune modulation. Sulfation is accomplished by the universal sulfate donor, PAPS (3'-Phosphoadenosine-5'-phosphosulfate), which is synthesized by bifunctional enzymes PAPSS1 and PAPSS2 (PAPS synthases). The PAPSS2 gene situates near PTEN and is frequently deleted with PTEN across cancer types. Approximately 20% of prostate cancer patients exhibit loss of PTEN, and ~50% of these cases also sustain a loss of PAPSS2. However, the loss of PAPSS2 appears to be tolerated and possibly compensated by its functionally redundant paralogue, PAPSS1, located on chromosome 4q24. The functional redundancy between PAPSS1 and PAPSS2 suggests that these two genes may be collateral lethality pair provided that sulfation is essential for cancer cell viability. Thus, we hypothesize that targeting PAPSS1 in PTEN/PAPSS2-null prostate cancer can generate cancer-specific vulnerabilities while leaving normal cells undisturbed. To assess this possibility, knockdown and knockout of PAPSS1 in cell lines of PAPSS2-null and PAPSS2-wildtype background were generated to characterize cell viability in vitro and tumor formation in vivo. PAPS and APS, an intermediary product of the sulfation pathway, are measured to verify that no alternative pathways for sulfate donors exist and that the co-extinction of PAPSS1/2 eliminates all avenues of generating sulfate donors. Combined extinction of PAPSS1/2 across multiple cancer cell lines was shown to be tolerated in vitro, and recurrent changes in morphology were observed. Loss of sulfation verified by the disappearance of sulfotyrosine and mass spectrometry measurements of PAPS and APS are pending. Our In vitro results surprisingly indicate that a major PTM, like sulfation is entirely dispensable for cancer cell viability under normal culture conditions. However, PAPSS1/2-null cell lines demonstrated a profound delay in tumor formation and prolonged survival, suggesting that sulfation may be required for stromal and innate immune modulation. Citation Format: Ko-Chien Chen, Yonhong Liu, Chenchu Lin, Er-Yen (Nick) Yen, Francesca Citron, Xingdi Ma, Tan Lin, Philip Lorenzi, Florian Muller, Ronald DePinho. Sulfation is required for prostate cancer xenograft tumor formation but is dispensable for cell viabilityin vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6053.

Defining tumor cell immune evasion Mouse models used to study cancer often lack a full immune system, allowing implantation of human tumors into the mice. By contrast, naturally evolving tumors must contend with a fully functional immune system and its destruction of some of the cells (see the Perspective by Ho and Wood). Two groups now report studies on mouse models with a fully intact immune system. Martin et al . started with preexisting murine tumor cell lines and examined their continued evolution in vivo, whereas Del Poggetto et al . examined the development of new pancreatic tumors in the context of inflammation, as is often seen in human patients. In each study, the authors found that the immune system exerted a selective pressure on cells that would give rise to tumors, promoting the survival of those that had lost expression of tumor suppressor genes or activated a specific oncogene. The findings suggest a major role for the immune system in driving tumor evolution across multiple types of cancer. —YN

We retrospectively collected a series of 82 endoscopically removed early colorectal cancers. Histological specimens were revised by two gastrointestinal pathologists, performing a re-evaluation of all risk factors for lymph node metastasis. The comparison between second opinion and first pathological report revealed that lymphovascular invasion and tumor grading showed a lower level of concordance than other parameters. Our results demonstrated that second opinion modified risk assessment in about 10% of cases. It was mainly due to a lack in reporting of some parameters at the first diagnosis and a different evaluation in second opinion for updated guidelines. Considering the subgroup of patients with modified risk assessment, clinical data revealed that tumors, re-classified as low risk, did not develop lymph node metastasis that, conversely, occurred in patients identified as high risk by second opinion. In conclusion, second opinion significantly alters risk perception of endoscopically removed early colorectal carcinomas representing a valuable tool for their appropriate clinical management.

The association between tumors and inflammation is a long-established clinical observation. Although many studies demonstrated that the inflammatory microenvironment can promote tumor growth through the activation of survival and proliferation programs in cancer cells, the reason why inflammation, an evolutionarily conserved response to damage aimed at reestablishing tissue integrity upon injury, might be integral to tumorigenesis still remains unknown. Pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by poor prognosis, represents a distinctive example of cooperation between inflammation and activated oncogenes. Frequently developed in a context of chronic pancreatitis, PDAC is always associated with an inflammatory microenvironment. As supported by a substantial body of evidence across a multitude of experimental models, when occurring in the context of pancreatitis, mutations of KRAS, the universal oncogenic driver of pancreatic cancer, lead to accelerated tumor development inducing the appearance of neoplastic precursor lesions, such as acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN), which can evolve into invasive tumors. Interestingly, preneoplastic pancreatic alterations, specifically ADM, have been previously identified in acute and chronic pancreatitis in the absence of oncogene activation. Investigating the effects of inflammation on normal pancreatic epithelial cells, we discovered that long after complete resolution a transient inflammatory event primes cells to cooperate with oncogenic KRAS. Indeed, upon recovery from a single acute inflammation, epithelial cells display an enduring adaptive response associated with sustained epigenetic and transcriptional reprogramming. Such adaptation facilitates the prompt reactivation of acinar-to-ductal metaplasia upon subsequent inflammatory events, representing a physiological mechanism for limiting tissue damage via rapid decrease of zymogen production. Because metaplastic lesions are mediated by the activation of MAPK signaling, we demonstrated that activating mutations of KRAS, maintaining an irreversible ADM, are protective against tissue damage in a contest of pancreatitis. Uncovering a new physiologic role of somatic oncogenic mutations in preserving tissue integrity during repeated damages, we propose that KRAS mutations represent a nearly universal event in pancreatic cancer because beneficial and under strong positive selection in the context of recurrent pancreatitis. Citation Format: I-Lin Ho, Edoardo Del Poggetto, Chiara Balestrieri, Er-Yen Yen, Francesca Citron, Rutvi Shah, Shaojun Zhang, Shan Jiang, Wantong Yao, Haoqiang Ying, Giannicola Genovese, Timothy Heffernan, Anirban Maitra, Linghua Wang, Timothy Wang, Giulio Draetta, Alessandro Carugo, Gioacchino Natoli, Andrea Viale. Epithelial memory of resolved inflammation cooperates with oncogenic KRAS to limit tissue damage while promoting pancreatic cancer [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 1327.

Simple Summary The possibility to identify, with appropriate biomarkers, patients that might mostly benefit from any given treatment is the basis of personalized oncology. Cancer biomarkers should be properly identified and validated on a large number of patients possibly enrolled in dedicated clinical trials. Here, we report the first molecular results of the MITO16A-ManGo-OV2 phase IV trial that was specifically designed to identify prognostic biomarkers of survival in epithelial ovarian cancer patients treated in first line with carboplatin-paclitaxel plus Bevacizumab (NCT01706120), a treatment for which validated predictive or prognostic biomarkers are still lacking. With this work we propose not only novel possible biomarkers for Bevacizumab-treated patients but also a way through which they can be properly collected, analyzed and statistically evaluated in the frame of large multicenter clinical trials. Abstract Background. Epithelial ovarian cancer (EOC) is a rare, highly lethal disease. In a subset of high grade EOC patients, maintenance therapy with the antiangiogenic drug Bevacizumab (BEV) is a valuable option. To date, no validated predictive or prognostic biomarkers exist for selecting EOC patients that might benefit from BEV treatment. Methods. Immunohistochemistry and RT-qPCR evaluated the expression of seven angiogenesis-related proteins and of a twelve microRNAs angio-signature in EOC patients, treated in first line with chemotherapy plus BEV (MITO16A/ManGO OV-2 phase IV trial). Centralized statistical analyses assessed the associations between each biomarker, clinical prognostic factors and survival outcomes. Results. High miR-484 expression was associated with longer progression-free and overall survival. Notably, the combined expression of miR-484 and its target VEGFB identified a subset of patients that might mostly benefit from BEV treatment. No other significant correlations were found between the other analyzed biomarkers and patients’ survival. The application of a shrinkage procedure to adjust for over-fitting hazard ratio estimates reduced the association significance. Conclusions. The analysis of angiogenesis related biomarkers in EOC patients homogenously treated with BEV in first line provides novel insight in their prognostic value and suggests that some of them might merit to be tested as predictive markers of drug activity in dedicated randomized trials.

Extensive efforts recently witnessed the complexity of cancer biology; however, molecular medicine still lacks the ability to elucidate hidden mechanisms for the maintenance of specific subclasses of rare tumors characterized by the silent onset and a poor prognosis (e.g., ovarian cancer, pancreatic cancer, and glioblastoma). Recent mutational fingerprints of human cancers highlighted genomic alteration occurring on epigenetic modulators. In this scenario, the epigenome dependency of cancer orchestrates a broad range of cellular processes critical for tumorigenesis and tumor progression, possibly mediating escaping mechanisms leading to drug resistance. Indeed, in this review, we discuss the pivotal role of chromatin remodeling in shaping the tumor architecture and modulating tumor fitness in a microenvironment-dependent context. We will also present recent advances in the epigenome targeting, posing a particular emphasis on how this knowledge could be translated into a feasible therapeutic approach to individualize clinical settings and improve patient outcomes.

The CDKN1B gene, encoding for the CDK inhibitor p27kip1, is mutated in defined human cancer subtypes, including breast, prostate carcinomas and small intestine neuroendocrine tumors. Lessons learned from small intestine neuroendocrine tumors suggest that CDKN1B mutations could be subclonal, raising the question of whether a deeper sequencing approach could lead to the identification of higher numbers of patients with mutations. Here, we addressed this question and analyzed human cancer biopsies from breast (n = 396), ovarian (n = 110) and head and neck squamous carcinoma (n = 202) patients, using an ultra‐deep sequencing approach. Notwithstanding this effort, the mutation rate of CDKN1B remained substantially aligned with values from the literature, showing that essentially only hormone receptor‐positive breast cancer displayed CDKN1B mutations in a relevant number of cases (3%). However, the analysis of copy number variation showed that another fraction of luminal breast cancer displayed loss (8%) or gain (6%) of the CDKN1B gene, further reinforcing the idea that the function of p27kip1 is important in this type of tumor. Intriguingly, an enrichment for CDKN1B alterations was found in samples from premenopausal luminal breast cancer patients (n = 227, 4%) and in circulating cell‐free DNA from metastatic luminal breast cancer patients (n = 59, 8.5%), suggesting that CDKN1B alterations could correlate with tumor aggressiveness and/or occur later during disease progression. Notably, many of the identified somatic mutations resulted in p27kip1 protein truncation, leading to loss of most of the protein or of its C‐terminal domain. Using a gene‐editing approach in a luminal breast cancer cell line, MCF‐7, we observed that the expression of p27kip1 truncating mutants that lose the C‐terminal domains failed to rescue most of the phenotypes induced by CDKN1B gene knockout, indicating that the functions retained by the C‐terminal portion are critical for its role as an oncosuppressor, at least in luminal breast cancer. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

miR-223 is an anti-inflammatory miRNA that in cancer acts either as an oncosuppressor or oncopromoter, in a context-dependent manner. In breast cancer, we demonstrated that it dampens the activation of the EGF pathway. However, little is known on the role of miR-223 during breast cancer onset and progression. miR-223 expression was decreased in breast cancer of luminal and HER2 subtypes and inversely correlated with patients' prognosis. In normal luminal mammary epithelial cells, miR-223 acted cell autonomously in the control of their growth and morphology in three-dimensional context. In the MMTV-Δ16HER2 transgenic mouse model, oncogene transformation resulted in a timely abrogation of miR-223 expression, likely due to activation of E2F1, a known repressor of miR-223 transcription. Accordingly, treatment with CDK4/6 inhibitors, which eventually results in restraining E2F1 activity, restored miR-223 expression and miR-223 ablation induced luminal breast cancer resistance to CDK4/6 inhibition, both in vitro and in vivo. Notably, miR-223 expression was lost in microdissected ductal carcinoma in situ (DCIS) from patients with luminal and HER2-positive breast cancer. Altogether, these results identify downmodulation of miR-223 as an early step in luminal breast cancer onset and suggest that it could be used to identify aggressive DCIS and predict the response to targeted therapy. Significance: miR-223 may represent a predictive biomarker of response to CDK4/6 inhibitors and its loss could identify DCIS lesions that are likely to progress into invasive breast cancer.

Epithelial Ovarian Cancer (EOC) is the most lethal gynecological cancer in developed countries, and the development of new strategies to overcome chemoresistance is an awaited clinical need. Angiogenesis, the development of new blood vessels from pre-existing vasculature, has been validated as a therapeutic target in this tumor type. The aim of this study is to verify if EOC cells with acquired resistance to platinum (PT) treatment display an altered angiogenic potential. Using a proteomic approach, we identified the tissue inhibitor of metalloproteinases 1 (TIMP-1) as the only secreted factor whose expression was up-regulated in PT-resistant TOV-112D and OVSAHO EOC cells used as study models. We report that TIMP-1 acts as a double-edged sword in the EOC microenvironment, directly affecting the response to PT treatment on tumor cells and indirectly altering migration and proliferation of endothelial cells. Interestingly, we found that high TIMP-1 levels in stage III&ndash, IV EOC patients associate with decreased overall survival, especially if they were treated with PT or bevacizumab. Taken together, these results pinpoint TIMP-1 as a key molecule involved in the regulation of EOC PT-resistance and progression disclosing the possibility that it could be used as a new biomarker of PT-resistance and/or therapeutic target.

This is a commentary to a paper and, as such, it has no abstract.

Postnatal development of the mammary gland relies on the maintenance of oriented cell division and apicobasal polarity, both of which are often deregulated in cancer. The microtubule (MT) network contributes to control these processes; however, very little is known about the impact of altered MT dynamics in the development of a complex organ and on the role played by MT-interacting proteins such as stathmin. In this study, we report that female stathmin knock-out (STM KO) mice are unable to nurse their litters due to frank impairment of mammary gland development. In mouse mammary epithelial cells, loss of stathmin compromised the trafficking of polarized proteins and the achievement of proper apicobasal polarity. In particular, prolactin receptor internalization and localization was altered in STM KO mammary epithelial cells, leading to decreased protein stability and downmodulation of the Prl/PrlR/STAT5 signaling pathway. Absence of stathmin induced alterations in mitotic spindle orientation, accumulation of mitotic defects, and apoptosis, overall contributing to tissue disorganization and further decreasing the expansion of the mammary epithelial compartment. Loss of stathmin in MMTV-Δ16HER2 transgenic mice decreased the incidence and increased the latency of these very aggressive mammary carcinomas. Collectively, these data identify the essential mammary protein stathmin as protumorigenic and suggest it may serve as a potential therapeutic target in breast cancer. Significance: Stathmin expression is critical to maintain oriented cell division and apicobasal polarity in normal mammary glands and to establish a protumorigenic program that eventually sustains HER2-positive breast cancer formation in mice.

Introduction. Most Head & Neck Squamous Cell Carcinoma (HNSCC) patients are diagnosed with a locally advanced disease. Radiotherapy (RT) plus anti-EGFR monoclonal antibodies (Cetuximab - CTX) represents an effective combination therapy for locally advanced HNSCC patients. However, the 5-year overall survival is still 45%, mainly due to the appearance of loco-regional recurrences, suggesting that the identification and validation of predictive biomarkers of CTX activity is urgently needed to identify patients at high-risk of recurrence, who will benefit more from this therapeutic strategy. We recently validated a 4-microRNAs (miRs) signature, related to Epithelial to Mesenchymal Transition (EMT) and able to stratify HNSCC patients at high-risk of recurrence development. Among these 4 miRs, miR-9 was the only up-regulated in primary tumors from patients who developed recurrence within 2-year follow-up. Here we evaluated whether miR-9 expression had a functional role in HNSCC onset, progression and response to therapies. Methods. miR-9 modified (overexpressing and silenced) HNSCC cells were generated, characterized for their growth and response to radio-, chemo- and targeted-therapies (i.e.Cisplatin, Paclitaxel, 5-Fluorouracil and CTX) in vitro and in vivo. Preclinical evidences were confirmed in a cohort of primary HNSCC samples by intercrossing the expression of miR-9 and selected target genes with patients’ clinical variables and response to therapy. Results. Biochemical and biological in vitro and in vivo experiments showed that high miR-9 expression triggers EMT and increases tumor initiating properties in HNSCC cells. Interestingly, miR-9 silenced HNSCC cells displayed a higher sensitivity to RT and CTX, but not to chemotherapy, when compared to controls. Using an in vivo model of HNSCC, we observed that intra-tumor injection of anti-miR-9 improves the efficacy of RT alone and remarkably in combination with CTX. Mechanistically, we demonstrated that in HNSCC cells, EGFR activation triggers miR-9 expression that promotes the transcription of SP1, establishing a positive forward loop between EGFR activation and SP1 transcription. Accordingly, in primary HNSCC miR-9 expression strongly correlates with the one of SP1 and EGFR. More importantly, in a cohort of HNSCC patients treated with RT plus CTX, high miR-9 expression acts as an efficient predictive biomarker of intrinsic resistance. Conclusion. Altogether by integrating wet-lab and clinical data, we provide strong evidences indicating that in HNSCC a subpopulation of miR-9 expressing cells is intrinsically resistant to RT plus CTX therapy, confirming its potential prognostic value. Since we set up an assay to easily and quantitatively evaluate miR-9 expression in diagnostic tumor biopsies, we propose that it could be used to personalize the treatments for this group of patients, avoiding unfaithful toxic therapies. Citation Format: Francesca Citron, Gian Luca Rampioni Vinciguerra, Giuseppe Fanetti, Ilenia Segatto, Barbara Belletti, Andrea Vecchione, Giovanni Franchin, Gustavo Baldassarre. miR-9 expression regulates and predicts the response to EGFR inhibitors in head & neck squamous cell carcinoma [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 3128.

PURPOSE: Head and Neck Squamous Cell Carcinomas (HNSCC) cause more than 300,000 deaths worldwide each year. Locoregional and distant recurrences represent worse prognostic events and accepted surrogate markers of patients’ overall survival. No valid biomarker and salvage therapy exist to identify and treat patients at high-risk of recurrence. We aimed to verify if selected microRNAs (miRs) could be used as biomarkers of recurrence in HNSCC. EXPERIMENTAL DESIGN: A Nanostring array was used to identify miRs associated with locoregional recurrence in 44 HNSCC patients. Bioinformatic approaches validated the signature and identified potential miR targets. Validation experiments were performed using an independent cohort of primary HNSCC samples and a panel of HNSCC cell lines. In vivo experiments validated the in vitro results. RESULTS: Our data identified a four-miR signature that classified HNSCC patients at high- or low-risk of recurrence. These miRs collectively impinge on the epithelial-mesenchymal transition process. In silico and wet lab approaches showed that miR-9, expressed at high levels in recurrent HNSCC, targets SASH1 and KRT13, while miR-1, miR-133 and miR-150, expressed at low levels in recurrent HNSCC, collectively target SP1 and TGFβ pathways. A six-genes signature comprising these targets identified patients at high risk of recurrences, as well. Combined pharmacological inhibition of SP1 and TGFβ pathways induced HNSCC cell death and, when timely administered, prevented recurrence formation in a preclinical model of HNSCC recurrence. CONCLUSIONS: By integrating different experimental approaches and competences, we identified critical mediators of recurrence formation in HNSCC that may merit to be considered for future clinical development. TRANSLATIONAL RELEVANCE. Most of HNSCC patients are diagnosed with a locally advanced disease and are treated with the combination of surgery, radiotherapy, and chemotherapy. This highly toxic approach is curative in about half of the cases but recurrent patients do not have effective salvage therapies. Therefore there is the urgency to identify and validate solid biomarkers able to classify patients at high risk that may benefit for specific targeted approaches. Our work tackled these two unmet clinical needs and identified a microRNA signature of locoregional recurrence in HNSCC patients. Starting from this signature, we identified two druggable pathways (i.e. SP1 and TGFβ) that when timely and concomitantly targeted efficiently prevented recurrence formation in a preclinical model. Both SP1 and TGFβ inhibitors have been already used to treat human patients, thus our work is of potential immediate translational relevance.

In breast cancer (BC) patients, local recurrences often arise in proximity of the surgical scar, suggesting that response to surgery may have a causative role. Radiotherapy (RT) after lumpectomy significantly reduces the risk of recurrence. We investigated the direct effects of surgery and of RT delivered intraoperatively (IORT), by collecting irradiated and non-irradiated breast tissues from BC patients, after tumor removal. These breast tissue specimens have been profiled for their microRNA (miR) expression, in search of differentially expressed miR among patients treated or not with IORT. Our results demonstrate that IORT elicits effects that go beyond the direct killing of residual tumor cells. IORT altered the wound response, inducing the expression of miR-223 in the peri-tumoral breast tissue. miR-223 downregulated the local expression of epidermal growth factor (EGF), leading to decreased activation of EGF receptor (EGFR) on target cells and, eventually, dampening a positive EGF-EGFR autocrine/paracrine stimulation loop induced by the post-surgical wound-healing response. Accordingly, both RT-induced miR-223 and peri-operative inhibition of EGFR efficiently prevented BC cell growth and reduced recurrence formation in mouse models of BC. Our study uncovers unknown effects of RT delivered on a wounded tissue and prompts to the use of anti-EGFR treatments, in a peri-operative treatment schedule, aimed to timely treat BC patients and restrain recurrence formation.

// Ilenia Pellizzari 1,* , Linda Fabris 1,4,* , Stefania Berton 1,* , Ilenia Segatto 1 , Francesca Citron 1 , Sara D’Andrea 1 , Martina Cusan 1 , Sara Benevol 1 , Tiziana Perin 2 , Samuele Massarut 3 , Vincenzo Canzonieri 2 , Monica Schiappacassi 1 , Barbara Belletti 1 and Gustavo Baldassarre 1 1 Division of Experimental Oncology 2, Department of Translational Research, CRO Aviano, National Cancer Institute, Aviano, Italy 2 Pathology Unit, CRO Aviano, National Cancer Institute, Aviano, Italy 3 Breast Surgery Unit, CRO Aviano, National Cancer Institute, Aviano, Italy 4 Department of Experimental Therapeutics, M.D. Anderson Cancer Center, Houston, TX, USA * These authors have contributed equally to this article Correspondence to: Gustavo Baldassarre, email: // Barbara Belletti, email: // Keywords : p27kip1; stathmin; H-Ras and K-Ras; cell cycle progression; metastasis Received : June 15, 2016 Accepted : July 19, 2016 Published : August 27, 2016 Abstract The tumor suppressor protein p27 Kip1 plays a pivotal role in the control of cell growth and metastasis formation. Several studies pointed to different roles for p27 Kip1 in the control of Ras induced transformation, although no explanation has been provided to elucidate these differences. We recently demonstrated that p27 kip1 regulates H-Ras activity via its interaction with stathmin. Here, using in vitro and in vivo models, we show that p27 kip1 is an important regulator of Ras induced transformation. In H-Ras V12 transformed cells, p27 kip1 suppressed cell proliferation and tumor growth via two distinct mechanisms: 1) inhibition of CDK activity and 2) impairment of MT-destabilizing activity of stathmin. Conversely, in K-Ras4B V12 transformed cells, p27 kip1 acted mainly in a CDK-dependent but stathmin-independent manner. Using human cancer-derived cell lines and primary breast and sarcoma samples, we confirmed in human models what we observed in mice. Overall, we highlight a pathway, conserved from mouse to human, important in the regulation of H-Ras oncogenic activity that could have therapeutic and diagnostic implication in patients that may benefit from anti-H-Ras therapies.

Despite novel treatment strategies and prevention, one of the major clinical challenges in breast cancer (BC) is the development of local and distant recurrences. These are linked not only to genetic and epigenetic alteration in cancer cells, but also to tumor-microenvironment and tumor-host crosstalk. In this context, surgical removal of primary tumor, inducing an inflammatory response and a wound healing process, may stimulate residual cancer cell survival, growth, and invasion. Radiotherapy, representing a standard care after surgery in BC, exerts its function by killing residual tumor cells and altering the local microenvironment. We have recently demonstrated that intraoperative radiotherapy strongly induced miR-223 expression that, by targeting EGF released in the peritumoral context, dampened the activation of EGFR/HER2 pathway, eventually resulting as a potent restrainer of residual cancer cell survival and growth. We also observed that miR-223 is highly expressed in normal mammary tissue, while all BC specimens analyzed expressed remarkably low levels. In line with these results, normal mammary epithelial cells of basal (HMEC) or luminal (BPEC) subtype expressed high levels of miR-223 compared to all tested BC cell lines. These data supported the possibility that loss of miR-223 expression could represent a common and possibly early event during mammary epithelial cell transformation. To explore this possibility, we transduced HMEC and BPEC cells using several oncogenes and tested the expression of miR-223. Transient expression of HER2, vSrc, and KRasG12D resulted in immediate drop of miR-223. Furthermore, to mimic oncogene-driven miR-223 down-modulation, we silenced its expression in HMEC and BPEC cells. Intriguingly, the loss of miR-223 per se induced the acquisition of transformed-like phenotypes, such as an increased proliferation in 2D-culture and a loss of polarized acinar structure with the formation of disorganized colonies in 3D-context. Interrogation of the TCGA BC dataset, comprising a large panel of normal and BC samples, confirmed our data and showed that, among all BC subtypes, miR-223 was particularly decreased in HER2+ and luminal BC. Notably, clustering HER2+ BC for miR-223HIGH/pY1068-EGFRLOW stratified patients with a significantly better overall survival. We thus explored the contribution of miR-223 in HER2-driven cell transformation. We overexpressed miR-223 in normal mammary epithelial cells and transformed them with HER2WT, ∆16HER2 (a particularly aggressive alternative splicing form of HER2), or HER2V659E (constitutively active form of HER2). Overexpression of miR-223 was able to efficiently counteract HER2-driven transformation, both in in vitro and in vivo models, not only in the presence of the WT oncogene, but also in the presence of the two aggressive mutants, the ∆16HER2 and HER2V659E. Altogether, our results indicate that loss or decreased expression of miR-223 may represent an early and common event during mammary epithelial cell transformation, contributing to loss of polarity and altering the maintenance of the differentiate phenotype, features profoundly linked to the tumorigenic process. Discovering the molecular mechanism by which miR-223 (and/or its targets) exerts their functions in cellular transformation and their possible interference in the tumor/stroma crosstalk may lead to identification of novel prognostic markers for early lesions and new promising therapeutic targets. Citation Format: Francesca Citron, Ilenia Segatto, Gian Luca Rampioni Vinciguerra, Gustavo Baldassarre, Barbara Belletti. Exploring the role of miR-223 loss in mammary epithelial cell transformation [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A72.

Microtubule (MT) dynamics is essential to provide a correct apico-basal polarization of epithelial cells, by regulating the movement of vesicles and proteins within the cell and by ensuring a correct orientation of the mitotic spindle during cell division. Little is known about the role of the MT-dynamics and of MT-regulating proteins during development of the mammary gland, a dynamic organ that displays a complex and finely organized apico-basal architecture. Stathmin, a MT-destabilizing protein, is often overexpressed in breast cancer (BC). By studying knockout (STM KO) mice we observed that STM KO females were unable to nurse their offspring. This phenotype was accompanied by defective development of the mammary gland, particularly evident during pregnancy and post-partum phases. Our results showed that stathmin absence mainly impinged on two key features of the mammary epithelial organization, polarity and proliferation. In vitro, normal mammary epithelial cells silenced for stathmin were not able to form organized acini in 3D-matrices. In vivo, when stathmin was KO, many molecules involved in mammary gland functions were not properly localized, indicating a disruption of the apico-basal polarity. Furthermore, loss of stathmin strongly decreased proliferation and induced disoriented positioning of the mitotic spindle in dividing epithelial cells, preventing the correct orientation of the daughter cells. Loss of polarity and deregulated proliferation are tightly linked to tumorigenesis. We therefore examined the role of stathmin also in the context of HER2-driven tumorigenesis. To this end, we used a transgenic mouse model expressing a constitutively active form of HER2 (Δ16HER2), which is an alternatively spliced, very aggressive form of HER2, and intercrossed them with STM WT and KO mice. We analyzed the role of stathmin in both early and late stages of tumorigenesis. STM KO mice displayed a significantly reduced number of pre-neoplastic foci and this phenotype was maintained also in late stages of tumorigenesis. In WT mice, pre-neoplastic lesions expressed high level of stathmin compared with normal tissue supporting the idea that stathmin played an active role in tumor initiation. Analysis of proliferation in tumors revealed that loss of stathmin strongly decreased the mitotic rate of Δ16HER2-transformed cells. Furthermore, we assessed that stathmin plays a cell autonomous function in tumor initiation. Our study has shed new light in the field of mammary gland development and of breast cancer biology. Collectively, our data indicate that loss of stathmin by altering the dynamics of microtubules, causes a profound disorganization of the normal mammary gland architecture and a delay in initiation of the tumorigenic process. We have uncovered new functions of stathmin that could have important implications in BC, also in consideration that several microtubules acting drugs are currently employed in the therapy of BC patients Citation Format: Ilenia Segatto, Mara De Marco Zompit, Gian Luca Rampioni Vinciguerra, Francesca Citron, Sara D'Andrea, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. Stathmin regulates mammary gland morphogenesis and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1460.