A comprehensive luminal breast cancer patient-derived xenografts (PDX) library to capture tumor heterogeneity and explore the mechanisms of resistance to CDK4/6 inhibitors.

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.

The Global Dam Watch database of river barrier and reservoir information for large-scale applications.

There are millions of river barriers worldwide, ranging from wooden locks to concrete dams, many of which form associated impoundments to store water in small ponds or large reservoirs. Besides their benefits, there is growing recognition of important environmental and social trade-offs related to these artificial structures. However, global datasets describing their characteristics and geographical distribution are often biased towards particular regions or specific applications, such as hydropower dams affecting fish migration, and are thus not globally consistent. Here, we present a new river barrier and reservoir database developed by the Global Dam Watch (GDW) consortium that integrates, harmonizes, and augments existing global datasets to support large-scale analyses. Data curation involved extensive quality control processes to create a single, globally consistent data repository of instream barriers and reservoirs that are co-registered to a digital river network. Version 1.0 of the GDW database contains 41,145 barrier locations and 35,295 associated reservoir polygons representing a cumulative storage capacity of 7,420 km3 and an artificial terrestrial surface water area of 304,600 km2.

Insights into the structural and functional activities of forgotten Kinases: PCTAIREs CDKs.

In cells, signal transduction heavily relies on the intricate regulation of protein kinases, which provide the fundamental framework for modulating most signaling pathways. Dysregulation of kinase activity has been implicated in numerous pathological conditions, particularly in cancer. The druggable nature of most kinases positions them into a focal point during the process of drug development. However, a significant challenge persists, as the role and biological function of nearly one third of human kinases remains largely unknown.Within this diverse landscape, cyclin-dependent kinases (CDKs) emerge as an intriguing molecular subgroup. In human, this kinase family encompasses 21 members, involved in several key biological processes. Remarkably, 13 of these CDKs belong to the category of understudied kinases, and only 5 having undergone broad investigation to date. This knowledge gap underscores the pressing need to delve into the study of these kinases, starting with a comprehensive review of the less-explored ones.Here, we will focus on the PCTAIRE subfamily of CDKs, which includes CDK16, CDK17, and CDK18, arguably among the most understudied CDKs members. To contextualize PCTAIREs within the spectrum of human pathophysiology, we conducted an exhaustive review of the existing literature and examined available databases. This approach resulted in an articulate depiction of these PCTAIREs, encompassing their expression patterns, 3D configurations, mechanisms of activation, and potential functions in normal tissues and in cancer.We propose that this effort offers the possibility of identifying promising areas of future research that extend from basic research to potential clinical and therapeutic applications.

Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer.

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.

Nutrient restriction-activated Fra-2 promotes tumor progression via IGF1R in miR-15a downmodulated pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease, characterized by an intense desmoplastic reaction that compresses blood vessels and limits nutrient supplies. PDAC aggressiveness largely relies on its extraordinary capability to thrive and progress in a challenging tumor microenvironment. Dysregulation of the onco-suppressor miR-15a has been extensively documented in PDAC. Here, we identified the transcription factor Fos-related antigen-2 (Fra-2) as a miR-15a target mediating the adaptive mechanism of PDAC to nutrient deprivation. We report that the IGF1 signaling pathway was enhanced in nutrient deprived PDAC cells and that Fra-2 and IGF1R were significantly overexpressed in miR-15a downmodulated PDAC patients. Mechanistically, we discovered that miR-15a repressed IGF1R expression via Fra-2 targeting. In miR-15a-low context, IGF1R hyperactivated mTOR, modulated the autophagic flux and sustained PDAC growth in nutrient deprivation. In a genetic mouse model, Mir15aKO PDAC showed Fra-2 and Igf1r upregulation and mTOR activation in response to diet restriction. Consistently, nutrient restriction improved the efficacy of IGF1R inhibition in a Fra-2 dependent manner. Overall, our results point to a crucial role of Fra-2 in the cellular stress response due to nutrient restriction typical of pancreatic cancer and support IGF1R as a promising and vulnerable target in miR-15a downmodulated PDAC.

Loss of the extracellular matrix glycoprotein EMILIN1 accelerates Δ16HER2-driven breast cancer initiation in mice.

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.

Mutant p53 sustains serine-glycine synthesis and essential amino acids intake promoting breast cancer growth.

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.

Impacts of existing and planned hydropower dams on river fragmentation in the Balkan Region.

The Balkan region has some of the best conserved rivers in Europe, but is also the location of ~3000 planned hydropower dams that are expected to help decarbonise energy production. A conflict between policies that promote renewable hydropower and those that prioritise river conservation has ensued, which can only be resolved with the help of reliable information. Using ground-truthed barrier data, we analysed the extent of current longitudinal river fragmentation in the Balkan region and simulated nine dam construction scenarios that varied depending on the number, location and size of the planned dams. Balkan rivers are currently fragmented by 83,017 barriers and have an average barrier density of 0.33 barriers/km after correcting for barrier underreporting; this is 2.2 times lower than the mean barrier density found across Europe and serves to highlight the relatively unfragmented nature of these rivers. However, our analysis shows that all simulated dam construction scenarios would result in a significant loss of connectivity compared to existing conditions. The largest loss of connectivity (-47 %), measured as reduction in barrier-free length, would occur if all planned dams were built, 20 % of which would impact on protected areas. The smallest loss of connectivity (-8 %) would result if only large dams (>10 MW) were built. In contrast, building only small dams (<10 MW) would cause a 45 % loss of connectivity while only contributing 32 % to future hydropower capacity. Hence, the construction of many small hydropower plants will cause a disproportionately large increase in fragmentation that will not be accompanied by a corresponding increase in hydropower. At present, hydropower development in the Balkan rivers does not require Strategic Environmental Assessment, and does not consider cumulative impacts. We encourage planners and policy makers to explicitly consider trade-offs between gains in hydropower and losses in river connectivity at the river basin scale.

HER2-Displaying M13 Bacteriophages induce Therapeutic Immunity against Breast Cancer.

The advent of trastuzumab has significantly improved the prognosis of HER2-positive (HER2+) breast cancer patients; nevertheless, drug resistance limits its clinical benefit. Anti-HER2 active immunotherapy represents an attractive alternative strategy, but effective immunization needs to overcome the patient's immune tolerance against the self-HER2. Phage display technology, taking advantage of phage intrinsic immunogenicity, permits one to generate effective cancer vaccines able to break immune tolerance to self-antigens. In this study, we demonstrate that both preventive and therapeutic vaccination with M13 bacteriophages, displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 or its Δ16HER2 splice variant on their surface (ECTM and Δ16ECTM phages), delayed mammary tumor onset and reduced tumor growth rate and multiplicity in ∆16HER2 transgenic mice, which are tolerant to human ∆16HER2. This antitumor protection correlated with anti-HER2 antibody production. The molecular mechanisms underlying the anticancer effect of vaccine-elicited anti-HER2 antibodies were analyzed in vitro against BT-474 human breast cancer cells, sensitive or resistant to trastuzumab. Immunoglobulins (IgG) purified from immune sera reduced cell viability mainly by impairing ERK phosphorylation and reactivating retinoblastoma protein function in both trastuzumab-sensitive and -resistant BT-474 cells. In conclusion, we demonstrated that phage-based HER2 vaccines impair mammary cancer onset and progression, opening new perspectives for HER2+ breast cancer treatment.

CDK4/6 Inhibitors in Combination Therapies: Better in Company Than Alone: A Mini Review.

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.

HMGA1 positively regulates the microtubule-destabilizing protein stathmin promoting motility in TNBC cells and decreasing tumour sensitivity to paclitaxel.

High Mobility Group A1 (HMGA1) is an architectural chromatin factor involved in the regulation of gene expression and a master regulator in Triple Negative Breast Cancer (TNBC). In TNBC, HMGA1 is overexpressed and coordinates a gene network that controls cellular processes involved in tumour development, progression, and metastasis formation. Here, we find that the expression of HMGA1 and of the microtubule-destabilizing protein stathmin correlates in breast cancer (BC) patients. We demonstrate that HMGA1 depletion leads to a downregulation of stathmin expression and activity on microtubules resulting in decreased TNBC cell motility. We show that this pathway is mediated by the cyclin-dependent kinase inhibitor p27kip1 (p27). Indeed, the silencing of HMGA1 expression in TNBC cells results both in an increased p27 protein stability and p27-stathmin binding. When the expression of both HMGA1 and p27 is silenced, we observe a significant rescue in cell motility. These data, obtained in cellular models, were validated in BC patients. In fact, we find that patients with high levels of both HMGA1 and stathmin and low levels of p27 have a statistically significant lower survival probability in terms of relapse-free survival (RFS) and distant metastasis-free survival (DMFS) with respect to the patient group with low HMGA1, low stathmin, and high p27 expression levels. Finally, we show in an in vivo xenograft model that depletion of HMGA1 chemo-sensitizes tumour cells to paclitaxel, a drug that is commonly used in TNBC treatments. This study unveils a new interaction among HMGA1, p27, and stathmin that is critical in BC cell migration. Moreover, our data suggest that taxol-based treatments may be more effective in reducing the tumour burden when tumour cells express low levels of HMGA1.

p27kip1 expression and phosphorylation dictate Palbociclib sensitivity in KRAS-mutated colorectal cancer.

In colorectal cancer, mutation of KRAS (RASMUT) 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 p27kip1 in the response to the selective CDK4/6-inhibitor Palbociclib, in colorectal cancer. Our results show that p27kip1 expression inversely correlated with Palbociclib response, both in vitro and in vivo. Generating a model of Palbociclib-resistant RASMUT colorectal cancer cells, we observed an increased expression of p27kip1, cyclin D, CDK4 and CDK6, coupled with an increased association between p27kip1 and CDK4. Furthermore, Palbociclib-resistant cells showed increased Src-mediated phosphorylation of p27kip1 on tyrosine residues and low doses of Src inhibitors re-sensitized resistant cells to Palbociclib. Since p27kip1 showed variable expression in RASMUT colorectal cancer samples, our study supports the possibility that p27kip1 could serve as biomarker to stratify patients who might benefit from CDK4/6 inhibition, alone or in combination with Src inhibitors.

Inhibition of CDK4/6 as Therapeutic Approach for Ovarian Cancer Patients: Current Evidences and Future Perspectives.

Alterations in components of the cell-cycle machinery are present in essentially all tumor types. In particular, molecular alterations resulting in dysregulation of the G1 to S phase transition have been observed in almost all human tumors, including ovarian cancer. These alterations have been identified as potential therapeutic targets in several cancer types, thereby stimulating the development of small molecule inhibitors of the cyclin dependent kinases. Among these, CDK4 and CDK6 inhibitors confirmed in clinical trials that CDKs might indeed represent valid therapeutic targets in, at least some, types of cancer. CDK4 and CDK6 inhibitors are now used in clinic for the treatment of patients with estrogen receptor positive metastatic breast cancer and their clinical use is being tested in many other cancer types, alone or in combination with other agents. Here, we review the role of CDK4 and CDK6 complexes in ovarian cancer and propose the possible use of their inhibitors in the treatment of ovarian cancer patients with different types and stages of disease.

miR-9 modulates and predicts the response to radiotherapy and EGFR inhibition in HNSCC.

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.

The many facets of miR-223 in cancer: Oncosuppressor, oncogenic driver, therapeutic target, and biomarker of response.

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.

RNA splicing alteration in the response to platinum chemotherapy in ovarian cancer: A possible biomarker and therapeutic target.

Since its discovery, alternative splicing has been recognized as a powerful way for a cell to amplify the genetic information and for a living organism to adapt, evolve, and survive. We now know that a very high number of genes are regulated by alternative splicing and that alterations of splicing have been observed in different types of human diseases, including cancer. Here, we review the accumulating knowledge that links the regulation of alternative splicing to the response to chemotherapy, focusing our attention on ovarian cancer and platinum-based treatments. Moreover, we discuss how expanding information could be exploited to identify new possible biomarkers of platinum response, to better select patients, and/or to design new therapies able to overcome platinum resistance.

CDKN1B mutation and copy number variation are associated with tumor aggressiveness in luminal breast cancer.

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.