Antibody-peptide conjugates deliver covalent inhibitors blocking oncogenic cathepsins.

Cysteine cathepsins are a family of proteases that are relevant therapeutic targets for the treatment of different cancers and other diseases. However, no clinically approved drugs for these proteins exist, as their systemic inhibition can induce deleterious side effects. To address this problem, we developed a modular antibody-based platform for targeted drug delivery by conjugating non-natural peptide inhibitors (NNPIs) to antibodies. NNPIs were functionalized with reactive warheads for covalent inhibition, optimized with deep saturation mutagenesis and conjugated to antibodies to enable cell-type-specific delivery. Our antibody-peptide inhibitor conjugates specifically blocked the activity of cathepsins in different cancer cells, as well as osteoclasts, and showed therapeutic efficacy in vitro and in vivo. Overall, our approach allows for the rapid design of selective cathepsin inhibitors and can be generalized to inhibit a broad class of proteases in cancer and other diseases.

CellCharter reveals spatial cell niches associated with tissue remodeling and cell plasticity.

Tissues are organized in cellular niches, the composition and interactions of which can be investigated using spatial omics technologies. However, systematic analyses of tissue composition are challenged by the scale and diversity of the data. Here we present CellCharter, an algorithmic framework to identify, characterize, and compare cellular niches in spatially resolved datasets. CellCharter outperformed existing approaches and effectively identified cellular niches across datasets generated using different technologies, and comprising hundreds of samples and millions of cells. In multiple human lung cancer cohorts, CellCharter uncovered a cellular niche composed of tumor-associated neutrophil and cancer cells expressing markers of hypoxia and cell migration. This cancer cell state was spatially segregated from more proliferative tumor cell clusters and was associated with tumor-associated neutrophil infiltration and poor prognosis in independent patient cohorts. Overall, CellCharter enables systematic analyses across data types and technologies to decode the link between spatial tissue architectures and cell plasticity.

Metabolic dependencies of metastasis-initiating cells in female breast cancer.

Understanding the mechanisms that enable cancer cells to metastasize is essential in preventing cancer progression. Here we examine the metabolic adaptations of metastasis-initiating cells (MICs) in female breast cancer and how those shape their metastatic phenotype. We find that endogenous MICs depend on the oxidative tricarboxylic acid cycle and fatty acid usage. Sorting tumor cells based upon solely mitochondrial membrane potential or lipid storage is sufficient at identifying MICs. We further identify that mitochondrially-generated citrate is exported to the cytoplasm to yield acetyl-CoA, and this is crucial to maintaining heightened levels of H3K27ac in MICs. Blocking acetyl-CoA generating pathways or H3K27ac-specific epigenetic writers and readers reduces expression of epithelial-to-mesenchymal related genes, MIC frequency, and metastatic potential. Exogenous supplementation of a short chain carboxylic acid, acetate, increases MIC frequency and metastasis. In patient cohorts, we observe that higher expression of oxidative phosphorylation related genes is associated with reduced distant relapse-free survival. These data demonstrate that MICs specifically and precisely alter their metabolism to efficiently colonize distant organs.

The Tumor Microenvironment as a Driving Force of Breast Cancer Stem Cell Plasticity.

Tumor progression involves the co-evolution of transformed cells and the milieu in which they live and expand. Breast cancer stem cells (BCSCs) are a specialized subset of cells that sustain tumor growth and drive metastatic colonization. However, the cellular hierarchy in breast tumors is rather plastic, and the capacity to transition from one cell state to another depends not only on the intrinsic properties of transformed cells, but also on the interplay with their niches. It has become evident that the tumor microenvironment (TME) is a major player in regulating the BCSC phenotype and metastasis. The complexity of the TME is reflected in its number of players and in the interactions that they establish with each other. Multiple types of immune cells, stromal cells, and the extracellular matrix (ECM) form an intricate communication network with cancer cells, exert a highly selective pressure on the tumor, and provide supportive niches for BCSC expansion. A better understanding of the mechanisms regulating these interactions is crucial to develop strategies aimed at interfering with key BCSC niche factors, which may help reducing tumor heterogeneity and impair metastasis.

TGFBI modulates tumour hypoxia and promotes breast cancer metastasis.

Breast cancer metastasis is a complex process that depends not only on intrinsic characteristics of metastatic stem cells, but also on the particular microenvironment that supports their growth and modulates the plasticity of the system. In search for microenvironmental factors supporting cancer stem cell (CSC) growth and tumour progression to metastasis, we here investigated the role of the matricellular protein transforming growth factor beta induced (TGFBI) in breast cancer. We crossed the MMTV-PyMT model of mammary gland tumorigenesis with a TgfbiΔ/Δ mouse and studied the CSC content of the tumours. We performed RNAseq on wt and ko tumours, and analysed the tumour vasculature and the immune compartment by IHC and FACS. The source of TGFBI expression was determined by qPCR and by bone marrow transplantation experiments. Finally, we performed in silico analyses using the METABRIC cohort to assess the potential prognostic value of TGFBI. We observed that deletion of Tgfbi led to a dramatic decrease in CSC content and lung metastasis. Our results show that lack of TGFBI resulted in tumour vessel normalisation, with improved vessel perfusion and decreased hypoxia, a major factor controlling CSCs and metastasis. Furthermore, human data mining in a cohort of breast cancer patients showed that higher expression of TGFBI correlates with poor prognosis and is associated with the more aggressive subtypes of breast cancer. Overall, these data reveal a novel biological mechanism controlling metastasis that could potentially be exploited to improve the efficacy and delivery of chemotherapeutic agents in breast cancer.

An Intrasplenic Injection Model for the Study of Cancer Stem Cell Seeding Capacity.

In many tumor types, only a minor pool of cancer cells-the so-called cancer stem cells-is able to colonize distant organs and give rise to secondary tumors. In humans, the liver is one of the main target organs for many metastatic tumor types, including colorectal cancer. However, mouse tumour models only rarely spontaneously metastasize to the liver. Therefore, reliable in vivo experimental metastasis assays are crucial to study cell seeding capacity and the mechanisms controlling these metastatic stem cell properties. Here, we describe an intrasplenic injection model that mimics the process of liver metastasis occurring in cancer patients.

MAGI1, a New Potential Tumor Suppressor Gene in Estrogen Receptor Positive Breast Cancer.

Membrane-associated guanylate kinase (MAGUK) with inverted domain structure-1 (MAGI1) is an intracellular adaptor protein that stabilizes epithelial junctions consistent with a tumor suppressive function in several cancers of epithelial origin. Here we report, based on experimental results and human breast cancer (BC) patients' gene expression data, that MAGI1 is highly expressed and acts as tumor suppressor in estrogen receptor (ER)+/HER2- but not in HER2+ or triple negative breast cancer (TNBC). Within the ER+/HER2- subset, high MAGI1 expression associates with ESR1 and luminal genes GATA3 and FOXA1 expression and better prognosis, while low MAGI1 levels correlates with higher histological grade, more aggressive phenotype and worse prognosis. Experimentally, MAGI1 downregulation in the ER+ human BC cells MCF7 impairs ER expression and signaling, promotes cell proliferation, and reduces apoptosis and epithelial differentiation. MAGI1 downregulation in the ER+ murine BC cell line 67NR accelerates primary tumor growth and enhances experimental lung metastasis formation. MAGI1 expression is upregulated by estrogen/ER, downregulated by prostaglandin E2/COX-2axis, and negatively correlates with inflammation in ER+/HER2- BC patients. Taken together, we show that MAGI1 is a new potential tumor suppressor in ER+/HER2- breast cancer with possible prognostic value for the identification of patients at high-risk of relapse within this subset.

Breast Cancer Stem Cells with Tumor- versus Metastasis-Initiating Capacities Are Modulated by TGFBR1 Inhibition.

Cancer stem cells (CSCs) are defined by their ability to regenerate a tumor upon transplantation. However, it is not yet clear whether tumors contain a single CSC population or different subsets of cells with mixed capacities for initiating primary and secondary tumors. Using two different identification strategies, we studied the overlap between metastatic stem cells and tumor-initiating cells (TICs) in the MMTV-PyMT model. Our results show that in the MMTV-PyMT model, Lin-CD90-ALDHhigh cells retained a high tumor-initiating potential (TIP) in orthotopic transplants, in contrast to Lin-CD24+CD90+, which retained higher metastatic capacity. Interestingly, suppression of TGFß signaling increased TIC numbers. We here describe the existence of distinct populations of CSCs with differing capacities to initiate tumors in the primary or the secondary site. Inhibiting TGFß signaling shifts the balance toward the former, which may have unanticipated implications for the therapeutic use of TGFß/TGFBR1 inhibitors.

Obesity promotes the expansion of metastasis-initiating cells in breast cancer.

BACKGROUND: Obesity is a strong predictor of poor prognosis in breast cancer, especially in postmenopausal women. In particular, tumors in obese patients tend to seed more distant metastases, although the biology behind this observation remains poorly understood. METHODS: To elucidate the effects of the obese microenvironment on metastatic spread, we ovariectomized C57BL/6 J female mice and fed them either a regular diet (RD) or a high-fat diet (HFD) to generate a postmenopausal diet-induced obesity model. We then studied tumor progression to metastasis of Py230 and EO771 grafts. We analyzed and phenotyped the RD and HFD tumors and the surrounding adipose tissue by flow cytometry, qPCR, immunohistochemistry (IHC) and western blot. The influence of the microenvironment on tumor cells was assessed by performing cross-transplantation of RD and HFD tumor cells into other RD and HFD mice. The results were analyzed using the unpaired Student t test when comparing two variables, otherwise we used one-way or two-way analysis of variance. The relationship between two variables was calculated using correlation coefficients. RESULTS: Our results show that tumors in obese mice grow faster, are also less vascularized, more hypoxic, of higher grade and enriched in CD11b+Ly6G+ neutrophils. Collectively, this favors induction of the epithelial-to-mesenchymal transition and progression to claudin-low breast cancer, a subtype of triple-negative breast cancer that is enriched in cancer stem cells. Interestingly, transplanting HFD-derived tumor cells in RD mice transfers enhanced tumor growth and lung metastasis formation. CONCLUSIONS: These data indicate that a pro-metastatic effect of obesity is acquired by the tumor cells in the primary tumor independently of the microenvironment of the secondary site. Effects of postmenopausal obesity on primary breast cancer tumoursᅟ.

Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis.

Integrins play an important role in haematopoietic stem cell (HSC) maintenance in the bone marrow niche. Here, we demonstrate that Periostin (Postn) via interaction with Integrin-αv (Itgav) regulates HSC proliferation. Systemic deletion of Postn results in peripheral blood (PB) anaemia, myelomonocytosis and lymphopenia, while the number of phenotypic HSCs increases in the bone marrow. Postn-/- mice recover faster from radiation injury with concomitant loss of primitive HSCs. HSCs from Postn-/- mice show accumulation of DNA damage generally associated with aged HSCs. Itgav deletion in the haematopoietic system leads to a similar PB phenotype and HSC-intrinsic repopulation defects. Unaffected by Postn, Vav-Itgav-/- HSCs proliferate faster in vitro, illustrating the importance of Postn-Itgav interaction. Finally, the Postn-Itgav interaction inhibits the FAK/PI3K/AKT pathway in HSCs, leading to increase in p27Kip1 expression resulting in improved maintenance of quiescent HSCs. Together, we demonstrate a role for Itgav-mediated outside-in signalling in regulation of HSC proliferation and stemness.

Interactions between cancer stem cells and their niche govern metastatic colonization.

Metastatic growth in distant organs is the major cause of cancer mortality. The development of metastasis is a multistage process with several rate-limiting steps. Although dissemination of tumour cells seems to be an early and frequent event, the successful initiation of metastatic growth, a process termed 'metastatic colonization', is inefficient for many cancer types and is accomplished only by a minority of cancer cells that reach distant sites. Prevalent target sites are characteristic of many tumour entities, suggesting that inadequate support by distant tissues contributes to the inefficiency of the metastatic process. Here we show that a small population of cancer stem cells is critical for metastatic colonization, that is, the initial expansion of cancer cells at the secondary site, and that stromal niche signals are crucial to this expansion process. We find that periostin (POSTN), a component of the extracellular matrix, is expressed by fibroblasts in the normal tissue and in the stroma of the primary tumour. Infiltrating tumour cells need to induce stromal POSTN expression in the secondary target organ (in this case lung) to initiate colonization. POSTN is required to allow cancer stem cell maintenance, and blocking its function prevents metastasis. POSTN recruits Wnt ligands and thereby increases Wnt signalling in cancer stem cells. We suggest that the education of stromal cells by infiltrating tumour cells is an important step in metastatic colonization and that preventing de novo niche formation may be a novel strategy for the treatment of metastatic disease.

Human SHBG mRNA translation is modulated by alternative 5'-non-coding exons 1A and 1B.

BACKGROUND: The human sex hormone-binding globulin (SHBG) gene comprises at least 6 different transcription units (TU-1, -1A, -1B, -1C, -1D and -1E), and is regulated by no less than 6 different promoters. The best characterized are TU-1 and TU-1A: TU-1 is responsible for producing plasma SHBG, while TU-1A is transcribed and translated in the testis. Transcription of the recently described TU-1B, -1C, and -1D has been demonstrated in human prostate tissue and prostate cancer cell lines, as well as in other human cell lines such as HeLa, HepG2, HeK 293, CW 9019 and imr 32. However, there are no reported data demonstrating their translation. In the present study, we aimed to determine whether TU-1A and TU-1B are indeed translated in the human prostate and whether 5' UTR exons 1A and 1B differently regulate SHBG translation. RESULTS: Cis-regulatory elements that could potentially regulate translation were identified within the 5'UTRs of SHBG TU-1A and TU-1B. Although full-length SHBG TU-1A and TU-1B mRNAs were present in prostate cancer cell lines, the endogenous SHBG protein was not detected by western blot in any of them. LNCaP prostate cancer cells transfected with several SHBG constructs containing exons 2 to 8 but lacking the 5'UTR sequence did show SHBG translation, whereas inclusion of the 5'UTR sequences of either exon 1A or 1B caused a dramatic decrease in SHBG protein levels. The molecular weight of SHBG did not vary between cells transfected with constructs with or without the 5'UTR sequence, thus confirming that the first in-frame ATG of exon 2 is the translation start site of TU-1A and TU-1B. CONCLUSIONS: The use of alternative SHBG first exons 1A and 1B differentially inhibits translation from the ATG situated in exon 2, which codes for methionine 30 of transcripts that begin with the exon 1 sequence.

Identification, characterization and expression of novel Sex Hormone Binding Globulin alternative first exons in the human prostate.

BACKGROUND: The human Sex Hormone Binding Globulin (SHBG) gene, located at 17p13.1, comprises, at least, two different transcription units regulated by two different promoters. The first transcription unit begins with the exon 1 sequence and is responsible for the production of plasma SHBG by the hepatocytes, while the second begins with an alternative exon 1 sequence, which replaces the exon 1 present in liver transcripts. Alternative exon 1 transcription and translation has only been demonstrated in the testis of transgenic mice containing an 11-kb human SHBG transgene and in the human testis. Our goal has been to further characterize the 5' end of the SHBG gene and analyze the presence of the SHBG alternative transcripts in human prostate tissue and derived cell lines. RESULTS: Using a combination of in silico and in vitro studies, we have demonstrated that the SHBG gene, along with exon 1 and alternative exon 1 (renamed here exon 1A), contains four additional alternative first exons: the novel exons 1B, 1C, and 1E, and a previously identified exon 1N, which has been further characterized and renamed as exon 1D. We have shown that these four alternative first exons are all spliced to the same 3' splice site of SHBG exon 2, and that exon 1A and the novel exon 1B can be spliced to exon 1. We have also demonstrated the presence of SHBG transcripts beginning with exons 1B, 1C and 1D in prostate tissues and cell lines, as well as in several non-prostatic cell lines. Finally, the alignment of the SHBG mammalian sequences revealed that, while exons 1C, 1D and 1E are very well conserved phylogenetically through non-primate mammal species, exon 1B probably aroused in apes due to a single nucleotide change that generated a new 5' splice site in exon 1B. CONCLUSION: The identification of multiple transcription start sites (TSS) upstream of the annotated first exon of human SHBG, and the detection of the alternative transcripts in human prostate, concur with the prediction of the ENCODE (ENCyclopedia of DNA Elements) project, and suggest that the regulation of SHBG is much more complex than previously reported.

Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis.

Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45(-), CD81(+) and Sca-1(+)). We also demonstrated that SP clonal cells secrete transforming growth factor beta1 (TGF-beta1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-beta1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.

Estrogen receptor beta displays cell cycle-dependent expression and regulates the G1 phase through a non-genomic mechanism in prostate carcinoma cells.

BACKGROUND: It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ERbeta) remain elusive. METHODS: We have analyzed the levels of ERbeta1 and ERbeta2 throughout the cell cycle, as well as the mechanisms of action and the consequences of the over-expression of ERbeta1 in the human prostate cancer LNCaP cell line. RESULTS: Both ERbeta1 mRNA and protein expression increased from the G1 to the S phase and decreased before entering the G2/M phase, whereas ERbeta2 levels decreased during the S phase and increased in the G2/M phase. ERbeta1 protein was detected in both the nuclear and non-nuclear fractions, and ERbeta2 was found exclusively in the nucleus. Regarding the mechanisms of action, endogenous ERbeta was able to activate transcription via ERE during the S phase in a ligand-dependent manner, whereas no changes in AP1 and NFkappaB transactivation were observed after exposure to estradiol or the specific inhibitor ICI 182,780. Over-expression of either wild type ERbeta1 or ERbeta1 mutated in the DNA-binding domain caused an arrest in early G1. This arrest was accompanied by the interaction of over-expressed ERbeta1 with c-Jun N-terminal protein kinase 1 (JNK1) and a decrease in c-Jun phosphorylation and cyclin D1 expression. The administration of ICI impeded the JNK1-ERbeta1 interaction, increased c-Jun phosphorylation and cyclin D1 expression and allowed the cells to progress to late G1, where they became arrested. CONCLUSIONS: Our results demonstrate that, in LNCaP prostate cancer cells, both ERbeta isoforms are differentially expressed during the cell cycle and that ERbeta regulates the G1 phase by a non-genomic mechanism.