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Breast cancer (BC) is marked by significant genetic, morphological and clinical heterogeneity. To capture this heterogeneity and unravel the molecular mechanisms driving tumor progression and drug resistance, we established a comprehensive patient-derived xenograft (PDX) biobank, focusing particularly on luminal (estrogen receptor, ER+) and young premenopausal patients, for whom PDX models are currently scarce. Across all BC subtypes, our efforts resulted in an overall success rate of 17% (26 established PDX lines out of 151 total attempts), specifically 15% in luminal, 12% in human epidermal growth factor receptor 2 positive (HER2+) and 35% in triple negative BC. These PDX mirrored morphologic and genetic features of BC from which they originated, serving as a reliable tool to investigate drug resistance and test therapeutic strategies. We focused on understanding resistance to CDK4/6 inhibitors (CDK4/6i), which are crucial in the treatment of patients with advanced luminal BC. Treating a sensitive luminal BC PDX with the CDK4/6i palbociclib revealed that, despite initial tumor shrinkage, some tumors might eventually regrow under drug treatment. RNA sequencing, followed by gene set enrichment analyses, unveiled that these PDXs have become refractory to CDK4/6i, both at biological and molecular levels, displaying significant enrichment in proliferation pathways, such as MTORC1, E2F and MYC. Using organoids derived from these PDX (PDxO), we observed that acquisition of CDK4/6i resistance conferred cross-resistance to endocrine therapy and that targeting MTORC1 was a successful strategy to overcome CDK4/6i resistance. Considered together, these results indicate that our PDX models may serve as robust tools to elucidate the molecular basis of BC disease progression and, by providing the possibility to simultaneously test different therapies on the same tumor, to surmount treatment resistance. While this approach is of course not feasible in the clinic, its exploitation in PDX may expedite the identification and development of more successful therapies for patients with advanced luminal BC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Platinum (PT)-resistant Epithelial Ovarian Cancer (EOC) grows as a metastatic disease, disseminating in the abdomen and pelvis. Very few options are available for PT-resistant EOC patients, and little is known about how the acquisition of PT-resistance mediates the increased spreading capabilities of EOC. Here, using isogenic PT-resistant cells, genetic and pharmacological approaches, and patient-derived models, we report that Integrin α6 (ITGA6) is overexpressed by PT-resistant cells and is necessary to sustain EOC metastatic ability and adhesion-dependent PT-resistance. Using in vitro approaches, we showed that PT induces a positive loop that, by stimulating ITGA6 transcription and secretion, contributes to the formation of a pre-metastatic niche enabling EOC cells to disseminate. At molecular level, ITGA6 engagement regulates the production and availability of insulin-like growth factors (IGFs), over-stimulating the IGF1R pathway and upregulating Snail expression. In vitro data were recapitulated using in vivo models in which the targeting of ITGA6 prevents PT-resistant EOC dissemination and improves PT-activity, supporting ITGA6 as a promising druggable target for EOC patients., (© 2024. The Author(s).)

The extracellular matrix (ECM) is an important component of the tumor microenvironment and undergoes extensive remodeling during both initiation and progression of breast cancer (BC). EMILIN1 is an ECM glycoprotein, whose function has been linked to cancer and metastasis. However, EMILIN1 role during mammary gland and BC development has never been investigated. In silico and molecular analyses of human samples from normal mammary gland and BC showed that EMILIN1 expression was lower in tumors than in healthy mammary tissue and it predicted poor prognosis, particularly in HER2-positive BC. HER2+ BC accounts for 15-20% of all invasive BC and is characterized by high aggressiveness and poor prognosis. The Δ16HER2 isoform, a splice variant with very high oncogenic potential, is frequently expressed in HER2+ BC and correlates with metastatic disease. To elucidate the role of EMILIN1 in BC, we analyzed the phenotype of MMTV-Δ16HER2 transgenic mice, developing spontaneous multifocal mammary adenocarcinomas, crossed with EMILIN1 knock-out (KO) animals. We observed that Δ16HER2/EMILIN1 KO female mice exhibited an accelerated normal mammary gland development and a significantly anticipated appearance of palpable tumors (13.32 vs 15.28 weeks). This accelerated tumor initiation was corroborated by an increased number of tumor foci observed in mammary glands from Δ16HER2/EMILIN1 KO mice compared to the wild-type counterpart. Altogether our results underscore the centrality of ECM in the process of BC initiation and point to a role for EMILIN1 during normal mammary gland development and in protecting from HER2-driven breast tumorigenesis., (© 2024. The Author(s).)

Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent growth. These findings reveal vulnerabilities potentially exploitable for tackling breast tumors bearing missense TP53 mutations., (© 2023. Springer Nature Limited.)

The cyclin D-CDK4/6 complexes play a pivotal role in controlling the cell cycle. Deregulation in cyclin D-CDK4/6 pathway has been described in many types of cancer and it invariably leads to uncontrolled cell proliferation. Many efforts have been made to develop a target therapy able to inhibit CDK4/6 activity. To date, three selective CDK4/6 small inhibitors have been introduced in the clinic for the treatment of hormone positive advanced breast cancer patients, following the impressive results obtained in phase III clinical trials. However, since their approval, clinical evidences have demonstrated that about 30% of breast cancer is intrinsically resistant to CDK4/6 inhibitors and that prolonged treatment eventually leads to acquired resistance in many patients. So, on one hand, clinical and preclinical studies fully support to go beyond breast cancer and expand the use of CDK4/6 inhibitors in other tumor types; on the other hand, the question of primary and secondary resistance has to be taken into account, since it is now very clear that neoplastic cells rapidly develop adaptive strategies under treatment, eventually resulting in disease progression. Resistance mechanisms so far discovered involve both cell-cycle and non-cell-cycle related escape strategies. Full understanding is yet to be achieved but many different pathways that, if targeted, may lead to reversion of the resistant phenotype, have been already elucidated. Here, we aim to summarize the knowledge in this field, focusing on predictive biomarkers, to recognize intrinsically resistant tumors, and therapeutic strategies, to overcome acquired resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rampioni Vinciguerra, Sonego, Segatto, Dall’Acqua, Vecchione, Baldassarre and Belletti.)

Given their intrinsic pleiotropism, microRNAs (miR) play complex biological roles, in both normal and pathological conditions. Often the same miR can act as oncogene or oncosuppressor, depending on the biological process dysregulated in each specific tissue. miR-223 does not represent an exception to this rule and its functions greatly differ in different contexts. miR-223 has been widely studied in the hematopoietic compartment, where it plays a central role in innate immune response, regulating myeloid differentiation and granulocytes function. Accordingly, dysregulated expression of miR-223 has been associated to different inflammatory disorders and tumors arising from the immune compartment. Most carcinomas, breast cancer being the most studied, display loss of miR-223. However, in gastro-esophageal cancers miR-223 is frequently overexpressed and correlates with worse prognosis. A link between miR-223 and response to CDK4/6-inhibitors has been recently proposed, suggesting a role as biomarker of therapeutic response. The notion that one of the most commonly mutated protein in cancer, mutant p53, binds the promoter of miR-223 and suppresses its transcription, adds a further level of complexity to the full understanding of miR-223 in cancer. In this review, we will summarize the current knowledge on the molecular networks that alter or are altered by miR-223, in different cancer types. We will discuss if the times are ready for the exploitation of miR-223 as predictive biomarker of treatment response or, even, as therapeutic target, in specific settings. Finally, we will suggest which could be the next steps to be taken for a realistic clinical application of miR-223. This article is categorized under: RNA in Disease and Development > RNA in Disease., (© 2021 The Authors. WIREs RNA published by Wiley Periodicals LLC.)

In colorectal cancer, mutation of KRAS (RAS MUT ) reduces therapeutic options, negatively affecting prognosis of the patients. In this setting, administration of CDK4/6-inhibitors, alone or in combination with other drugs, is being tested as promising therapeutic strategy. Identifying sensitive patients and overcoming intrinsic and acquired resistance to CDK4/6 inhibition represent still open challenges, to obtain better clinical responses. Here, we investigated the role of the CDK inhibitor p27 kip1 in the response to the selective CDK4/6-inhibitor Palbociclib, in colorectal cancer. Our results show that p27 kip1 expression inversely correlated with Palbociclib response, both in vitro and in vivo. Generating a model of Palbociclib-resistant RAS MUT colorectal cancer cells, we observed an increased expression of p27 kip1 , cyclin D, CDK4 and CDK6, coupled with an increased association between p27 kip1 and CDK4. Furthermore, Palbociclib-resistant cells showed increased Src-mediated phosphorylation of p27 kip1 on tyrosine residues and low doses of Src inhibitors re-sensitized resistant cells to Palbociclib. Since p27 kip1 showed variable expression in RAS MUT colorectal cancer samples, our study supports the possibility that p27 kip1 could serve as biomarker to stratify patients who might benefit from CDK4/6 inhibition, alone or in combination with Src inhibitors., (© 2021. The Author(s).)

Radiotherapy (RT) plus the anti-EGFR monoclonal antibody Cetuximab (CTX) is an effective combination therapy for a subset of head and neck squamous cell carcinoma (HNSCC) patients. However, predictive markers of efficacy are missing, resulting in many patients treated with disappointing results and unnecessary toxicities. Here, we report that activation of EGFR upregulates miR-9 expression, which sustains the aggressiveness of HNSCC cells and protects from RT-induced cell death. Mechanistically, by targeting KLF5, miR-9 regulates the expression of the transcription factor Sp1 that, in turn, stimulates tumor growth and confers resistance to RT+CTX in vitro and in vivo. Intriguingly, high miR-9 levels have no effect on the sensitivity of HNSCC cells to cisplatin. In primary HNSCC, miR-9 expression correlated with Sp1 mRNA levels and high miR-9 expression predicted poor prognosis in patients treated with RT+CTX. Overall, we have discovered a new signaling axis linking EGFR activation to Sp1 expression that dictates the response to combination treatments in HNSCC. We propose that miR-9 may represent a valuable biomarker to select which HNSCC patients might benefit from RT+CTX therapy., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

The CDKN1B gene, encoding for the CDK inhibitor p27 kip1 , 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 p27 kip1 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 p27 kip1 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 p27 kip1 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., (© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.)

For many tumor types chemotherapy still represents the therapy of choice and many standard treatments are based on the use of platinum (PT) drugs. However, de novo or acquired resistance to platinum is frequent and leads to disease progression. In Epithelial Ovarian Cancer (EOC) patients, PT-resistant recurrences are very common and improving the response to treatment still represents an unmet clinical need. To identify new modulators of PT-sensitivity, we performed a loss-of-function screening targeting 680 genes potentially involved in the response of EOC cells to platinum. We found that SGK2 (Serum-and Glucocorticoid-inducible kinase 2) plays a key role in PT-response. We show here that EOC cells relay on the induction of autophagy to escape PT-induced death and that SGK2 inhibition increases PT sensitivity inducing a block in the autophagy cascade due to the impairment of lysosomal acidification. Mechanistically we demonstrate that SGK2 controls autophagy in a kinase-dependent manner by binding and inhibiting the V-ATPase proton pump. Accordingly, SGK2 phosphorylates the subunit V1H (ATP6V1H) of V-ATPase and silencing or chemical inhibition of SGK2, affects the normal autophagic flux and sensitizes EOC cells to platinum. Hence, we identified a new pathway that links autophagy to the survival of cancer cells under platinum treatment in which the druggable kinase SGK2 plays a central role. Our data suggest that blocking autophagy via SGK2 inhibition could represent a novel therapeutic strategy to improve patients' response to platinum.

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., (©2019 American Association for Cancer Research.)

The p27 kip1 protein, mainly known as a negative regulator of cell proliferation, has also been involved in the control of other cellular processes, including the regulation of cytoskeleton dynamics. Notably, these two functions involve distinct protein domains, residing in the N- and C-terminal halves, respectively. In the last two decades, p27 kip1 has been reported to interact with microtubule and acto-myosin cytoskeletons, both in direct and indirect ways, overall drawing a picture in which several factors play their role either in synergy or in contrast one with another. As a result, the role of p27 kip1 in cytoskeleton dynamics has been implicated in cell migration, both in physiologic and in neoplastic contexts, modulating cytokinesis, lipid raft trafficking, and neuronal development. Recently, two distinct papers have further reported a central role for p27 kip1 in the control of microtubule stability and post-translational modifications, dissecting the interaction between p27 kip1 and α-tubulin-acetyl-transferase (α-TAT), an enzyme involved in the stability of microtubules, and protein-regulator of cytokinesis 1 (PRC1), a nuclear regulator of the central spindle during mitosis. In light of these recent evidences, we will comment on the role of p27 kip1 on cytoskeleton regulation and its implication for cancer progression., Competing Interests: The authors declare that they have no competing interests.

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., (©2018 American Association for Cancer Research.)

Signal transducer and activator of transcription 3 (STAT3) is responsible for mediating the transcriptional programs downstream of several cytokine, growth factor, and oncogenic stimuli. Its expression and activity are consistently linked to cellular transformation, as well as tumor initiation and progression. Due to this central role, STAT3 is widely considered a good target for anti-cancer therapy; however, the success of these approaches has been, so far, very limited. Notably, on one side, STAT3 is aberrantly active in many breast cancers, on the other, at the physiological level, it is the main mediator of epithelial cell death during post-lactation mammary-gland involution, thus strongly suggesting that its biological functions are highly context-specific. One of the most peculiar features of STAT3 is that it can act both in cell-autonomous and non-cell-autonomous manners, simultaneously modulating the phenotypes of the tumor cells and their microenvironment. Here, we focus on the role of STAT3 in breast cancer progression, discussing the potential contrasting roles of STAT3 activation in the establishment of locally recurrent and distant metastatic disease. Based on the most recent literature, depending on the tumor cell type, the local microenvironment status, and the stage of the disease, either STAT3 activation or inactivation can support disease progression. Accordingly, cancer cells dynamically exploit STAT3 activity to carry out transcriptional programs somehow contrasting and complementary, such as supporting survival and growth, dormancy and awakening, stem cell-like features, and inflammation, immune response, and immune evasion. As a consequence, to achieve clinical efficacy, the conception and testing of anti-STAT3 targeted therapies will need a very careful evaluation of these opposing roles and of the most appropriate tumor context, disease stage and patient population to treat.

The CDKN1B gene encodes for the p27 Kip1 protein, firstly characterized as a cyclin dependent kinase (CDK)-inhibitor. Germline CDKN1B pathogenic variants have been described in hereditary tumors, such as multiple endocrine neoplasia (MEN)-like syndromes and familial prostate cancer. Despite its central role in tumor progression, for a long time it has been proposed that CDKN1B was very rarely somatically mutated in human cancer and that its expression levels were almost exclusively regulated at post-transcriptional level. Yet, the advent of massive parallel sequencing has partially subverted this general understanding demonstrating that, at least in some types of cancer, CDKN1B is mutated in a significant percentage of analyzed samples. Recent works have demonstrated that CDKN1B can be genetically inactivated and this occurs particularly in sporadic luminal breast cancer, prostate cancer and small intestine neuroendocrine tumors. However, a clear picture of the extent and significance of CDKN1B mutations in human malignances is still lacking. To fill this gap, we interrogated the COSMIC, ICGC, cBioPortal, and TRANSFAC data portals and current literature in PubMed, and reviewed the mutational spectrum of CDKN1B in human cancers, interpreting the possible impact of these mutations on p27 Kip1 protein function and tumor onset and progression.

Genomic instability represents a typical feature of aggressive cancers. Normal cells have evolved intricate responses to preserve genomic integrity in response to stress, such as DNA damage induced by γ-irradiation. Cyclin-dependent kinases (CDKs) take crucial part to these safeguard mechanisms, but involvement of CDK-inhibitors, such as p27 Kip1 , is less clear. We generated immortalized fibroblasts from p27 kip1 knock-out (KO) mouse embryos and re-expressed p27 kip1 WT, or its mutant forms, to identify the function of different domains. We γ-irradiated fibroblasts and observed that loss of p27 Kip1 was associated to accumulation of residual DNA damage, increased number of mitotic aberration and, eventually, to survival advantage. Nuclear localization and cyclin/CDK-binding of p27 Kip1 were critical to mediate proper response to DNA damage. In human luminal breast cancer (LBC) p27 kip1 is frequently down-modulated and CDKN1B, p27 Kip1 gene, sporadically mutated. We recapitulated results obtained in mouse fibroblasts in a LBC cell line genetically manipulated to be KO for CDKN1B gene. Following γ-irradiation, we confirmed that p27 kip1 expression was necessary to preserve genomic integrity and to recognize and clear-out aberrant cells. Our study provides important insights into mechanisms underlying radio-resistance and unveils the possibility for novel treatment options exploiting DNA repair defects in LBC.

The tumor suppressor protein p27Kip1 plays a pivotal role in the control of cell growth and metastasis formation.Several studies pointed to different roles for p27Kip1 in the control of Ras induced transformation, although no explanation has been provided to elucidate these differences. We recently demonstrated that p27kip1 regulates H-Ras activity via its interaction with stathmin.Here, using in vitro and in vivo models, we show that p27kip1 is an important regulator of Ras induced transformation. In H-RasV12 transformed cells, p27kip1 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-Ras4BV12 transformed cells, p27kip1 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.

Unlabelled: Breast cancer is a frequent and treatable disease. However, when recurrent, breast cancer often becomes refractory to therapy and progresses into metastatic forms that are typically incurable. Thus, understanding and targeting the critical pathways underlying breast cancer recurrence is urgently needed to eradicate primary disease and achieve better prognosis. Recently, we have demonstrated that the ribosomal protein p70S6K is activated in residual breast cancer cells as a result of post-surgical inflammation and that interfering with its activity in the peri-operative setting strongly suppresses recurrence in a mouse model. In order to develop clinically-exploitable treatments targeting p70S6K, we have tested a newly generated compound, called FS-115. FS-115 potently inhibited p70S6K1 (IC50 35nM) with high selectivity over other AGC kinases or PI3K pathway kinases. In vitro, treatment with FS-115 efficiently blocked p70S6K activity in breast cancer cell lines and impaired colony formation and anchorage independent growth. Pharmacokinetic profiling showed that FS-115 exhibited high oral bioavailability, optimal plasma distribution and high brain penetrance. In nude mice, FS-115 strongly suppressed tumor take-rate and primary tumor growth. Oral dosing with FS-115 in a peri-operative schedule was effective in decreasing local recurrence of breast cancer and a long-term treatment schedule was well tolerated and efficiently suppressed distant metastasis formation. Altogether, we propose that FS-115 might be a good candidate for the treatment of breast cancer patients at high risk to relapse., Summary Statement: Our results confirm that inhibition of p70S6K represents a valuable opportunity for restraining loco-regional relapse and metastasis in breast cancer and identify in FS-115 a promising candidate-inhibitor to move from preclinical to clinical treatments.

The cyclin-dependent kinase (CDK) inhibitor p27(kip1) is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27(kip1)-null mice is reverted by concomitant deletion of stathmin in p27(kip1)/stathmin double-KO mice, suggesting that a CDK-independent function of p27(kip1) contributes to the control of cell proliferation. Whether the regulation of MT stability by p27(kip1) impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27(kip1)-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27(kip1) and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27(kip1), by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27(kip1) and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway.

Inflammation is clinically linked to cancer but the mechanisms are not fully understood. Surgery itself elicits a range of inflammatory responses, suggesting that it could represent a perturbing factor in the process of local recurrence and/or metastasis. Post-surgery wound fluids (WF), drained from breast cancer patients, are rich in cytokines and growth factors, stimulate the in vitro growth of breast cancer cells and are potent activators of the STAT transcription factors. We wondered whether STAT signaling was functionally involved in the response of breast cancer cells to post-surgical inflammation. We discovered that WF induced the enrichment of breast cancer cells with stem-like phenotypes, via activation of STAT3. In vitro, WF highly stimulated mammosphere formation and self-renewal of breast cancer cells. In vivo, STAT3 signaling was critical for breast cancer cell tumorigenicity and for the formation of local relapse after surgery. Overall, we demonstrate here that surgery-induced inflammation promotes stem-like phenotypes and tumor-initiating abilities of breast cancer cells. Interfering with STAT3 signaling with a peri-surgical treatment was sufficient to strongly suppress this process. The understanding of the crosstalk between breast tumor-initiating cells and their microenvironment may open the way to successful targeting of these cells in their initial stages of growth and be eventually curative.

In early breast cancer, local relapses represent a determinant and not simply an indicator of risk for distant relapse and death. Notably, 90% of local recurrences occur at or close to the same quadrant of the primary cancer. Relevance of PI3K/mTOR/p70S6K signaling in breast tumorigenesis is very well documented. However, the pathway/s involved in the process of breast cancer local relapse are not well understood. The ribosomal protein p70S6K has been implicated in breast cancer cell response to post-surgical inflammation, supporting the hypothesis that it may be crucial also for breast cancer recurrence. Here, we show that p70S6K activity is required for the survival of breast cancer cells challenged in "hostile" microenvironments. We found that impairment of p70S6K activity in breast cancer cells strongly decreased their tumor take rate in nude mice. In line with this observation, if cells were challenged to grow in anchorage independence or in clonogenic assay, growth of colonies was strongly dependent on an intact p70S6K signaling. This in vitro finding was particularly evident when breast cancer cells were grown in the presence of wound fluids harvested following surgery from breast cancer patients, suggesting that the stimuli present in the post-surgical setting at least partially relied on activity of p70S6K to stimulate breast cancer relapse. From a mechanistic point of view, our results indicated that p70S6K signaling was able to activate Gli1 and up-regulate the anti-apoptotic protein Bcl2, thereby activating a survival response in breast cancer cells challenged in hostile settings. Our work highlights a previously poorly recognized function of p70S6K in preserving breast cancer cell survival, which could eventually be responsible for local relapse and opens the way to the design of new and more specific therapies aiming to restrain the deleterious effects of wound response., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

MicroRNAs (miRs) are a large class of small regulatory RNAs that function as nodes of signaling networks. This implicates that miRs expression has to be finely tuned, as observed during cell cycle progression. Here, using an expression profiling approach, we provide evidence that the CDK inhibitor p27Kip1 regulates miRs expression following cell cycle exit. By using wild type and p27KO cells harvested in different phases of the cell cycle we identified several miRs regulated by p27Kip1 during the G1 to S phase transition. Among these miRs, we identified miR-223 as a miR specifically upregulated by p27Kip1 in G1 arrested cells. Our data demonstrate that p27Kip1 regulated the expression of miR-223, via two distinct mechanisms. p27Kip1 directly stabilized mature miR-223 expression, acting as a RNA binding protein and it controlled E2F1 expression that, in turn, regulated miR-223 promoter activity. The resulting elevated miR-223 levels ultimately participated to arresting cell cycle progression following contact inhibition. Importantly, this mechanism of growth control was conserved in human cells and deranged in breast cancers. Here, we identify a novel and conserved function of p27Kip1 that, by modulating miR-223 expression, contributes to proper regulation of cell cycle exit following contact inhibition. Thus we propose a new role for miR-223 in the regulation of breast cancer progression.

The CDK inhibitor p27(kip1) is a critical regulator of cell cycle progression, but the mechanisms by which p27(kip1) controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner. To get more insights into the in vivo significance of this interaction, we generated p27(kip1) and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27(kip1) null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

The microtubule-destabilizing protein stathmin is highly expressed in several types of tumor, thus deserving the name of oncoprotein 18. High levels of stathmin expression and/or activity favor the metastatic spreading and mark the most aggressive tumors, thus representing a realistic marker of poor prognosis. Stathmin is a downstream target of many signaling pathways, including Ras-MAPK, PI3K and p53, involved in both tumor onset and progression. We thus hypothesized that stathmin could also play a role during the early stages of tumorigenesis, an issue completely unexplored. In order to establish whether stathmin expression is necessary for tumor initiation, we challenged wild type (WT), stathmin heterozygous and stathmin knock-out (KO) mice with different carcinogens. Using well-defined mouse models of carcinogenesis of skin, bladder and muscle by the means of 7,12-dimethylbenz[α]antracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA), N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) and 3-methylcholanthrylene (3MC) treatments, respectively, we demonstrated that knock-out of stathmin has no impact on the onset of cancer in mice. No significant difference was noticed either when the Ras oncogene was mutated (skin carcinogenesis model) or when the p53 pathway was inactivated (bladder carcinomas and fibrosarcomas). Finally, we concomitantly impinged on p53 and Ras pathways, by generating WT and stathmin KO mouse embryo fibroblasts transformed with papilloma virus large T antigen (LgTAg) plus the K-Ras(G12V) oncogene. In vivo growth of xenografts from these transformed fibroblasts did not highlight any significant difference depending on the presence or absence of stathmin. Overall, our work demonstrates that stathmin expression is dispensable for tumor onset, at least in mice, thus making stathmin a virtually exclusive marker of aggressive disease and a promising therapeutic target for advanced cancers.