PDF file - 79K, Expression level of molecules involved in E-cadherin down-regulation. Western blot analysis was performed using total cell lysates (A) or lysates from cytoplasmic or nuclear fraction (B). The indicated Abs were used. RT-PCR was performed to examine mRNA expression level of Axin2 in TE2 and HCE4 cells and GAPDH was used as an internal control

PDF file - 59K, PKCK2 activity dependent stabilization of exogenous Snail. A, MCF-7 cells that have low PKCK2 activity were co-transfected with Flag tagged Snail expression vector along either with empty vector or with Myc tagged PKCK2 expression vector. B, HCE4 cells were transfected with Flag-Snail and then were treated or un-treated with TBB, a pharmacologic inhibitor of PKCK2. C, PKCK2 activity was measured for each condition. S92 represents a vector expressing wild type snail, S92A (serine 92 replaced with alanine) mutant represents a vector expressing non-phosphorylatable form of snail, and S92E (serine 92 replaced with glutamic acid) represents a vector expressing mutant snail to mimic constitutive phosphorylation

PDF file - 71K, Western blot analysis for type of cadherin expressed in TE2, TE3, HCE4, and HCE7 (A) and for epithelial or mesenchymal markers and PKB/Akt activation in TE3 and HCE7 cells (B)

Abstract Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum‐containing media. The majority of researchers use serum‐containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non‐sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA‐MB‐231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano‐flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large‐scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.

Metastatic spread of a cancer to secondary sites is a coordinated, non-random process. Cancer cell-secreted vesicles, especially exosomes, have recently been implicated in the guidance of metastatic dissemination, with specific surface composition determining some aspects of organ-specific localization. Nevertheless, whether the tumor microenvironment influences exosome biodistribution has yet to be investigated. Here, we show that microenvironmental cytokines, particularly CCL2, decorate cancer exosomes via binding to surface glycosaminoglycan side chains of proteoglycans, causing exosome accumulation in specific cell subsets and organs. Exosome retention results in changes in the immune landscape within these organs, coupled with a higher metastatic burden. Strikingly, CCL2-decorated exosomes are directed to a subset of cells that express the CCL2 receptor CCR2, demonstrating that exosome-bound cytokines are a crucial determinant of exosome-cell interactions. In addition to the finding that cytokine-conjugated exosomes are detected in the blood of cancer patients, we discovered that healthy subjects derived exosomes are also associated with cytokines. Although displaying a different profile from exosomes isolated from cancer patients, it further indicates that specific combinations of cytokines bound to exosomes could likewise affect other physiological and disease settings.

Background Genome-wide association studies have identified 196 high confidence independent signals associated with breast cancer susceptibility. Variants within these signals frequently fall in distal regulatory DNA elements that control gene expression. Results We designed a Capture Hi-C array to enrich for chromatin interactions between the credible causal variants and target genes in six human mammary epithelial and breast cancer cell lines. We show that interacting regions are enriched for open chromatin, histone marks for active enhancers, and transcription factors relevant to breast biology. We exploit this comprehensive resource to identify candidate target genes at 139 independent breast cancer risk signals and explore the functional mechanism underlying altered risk at the 12q24 risk region. Conclusions Our results demonstrate the power of combining genetics, computational genomics, and molecular studies to rationalize the identification of key variants and candidate target genes at breast cancer GWAS signals.

Within the tumor microenvironment, there is an intricate communication happening between tumor and stromal cells. This information exchange, in the form of cytokines, growth factors, extracellular vesicles, danger molecules, cell debris, and other factors, is capable of modulating the function of immune cells. The triggering of specific responses, including phenotypic alterations, can ultimately result in either immune surveillance or tumor cell survival. Macrophages are a well-studied cell lineage illustrating the different cellular phenotypes possible, depending on the tumor microenvironmental context. While our understanding of macrophage responses is well documentedin vitro, surprisingly, little work has been done to confirm these observations in the cancer microenvironment. In fact, there are examples of opposing reactions of macrophages to cytokines in cell culture andin vivotumor settings. Additionally, it seems that different macrophage lineages, for example tissue-resident and monocyte-derived macrophages, respond differently to cytokines and other cancer-derived signals. In this review article, we will describe and discuss the diverging reports on how cancer cells influence monocyte-derived and tissue-resident macrophage traitsin vivo.

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes antitumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8(+) TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226(hi) TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226(+)CD8(+) T cells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.

Breast cancer genome-wide association studies (GWASs) have identified 150 genomic risk regions containing more than 13,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements. However, the genes underlying breast cancer risk associations are largely unknown. Here, we used genetic colocalization analysis to identify loci at which gene expression could potentially explain breast cancer risk phenotypes. Using data from the Breast Cancer Association Consortium (BCAC) and quantitative trait loci (QTL) from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Project (TCGA), we identify shared genetic relationships and reveal novel associations between cancer phenotypes and effector genes. Seventeen genes, including NTN4, were identified as potential mediators of breast cancer risk. For NTN4, we showed the rs61938093 CCV at this region was located within an enhancer element that physically interacts with the NTN4 promoter, and the risk allele reduced NTN4 promoter activity. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. These data provide evidence linking risk-associated variation to genes that may contribute to breast cancer predisposition.

Fine-mapping of breast cancer GWAS regions has identified 195 high confidence signals containing more than 5,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements and thus may confer the risk by altering gene expression in cis . We analyzed allelic expression imbalance (AEI) of genes surrounding known breast cancer signals, using normal breast and breast tumor transcriptome data and imputed genotypes. Fourteen genes, including NTN4 , were identified whose expression was associated with CCV genotype. We showed that CCVs at this signal were located within an enhancer that physically interacts with the NTN4 promoter. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo . Here, we present the most comprehensive AEI analysis of breast cancer CCVs resulting in identification of new candidate risk genes.

Naïve CD4+ T cells in the periphery differentiate into regulatory T cells (Tregs) in which Foxp3 is expressed for their suppressive function. NLRP3, a pro-inflammatory molecule, is known to be involved in inflammasome activation associated with several diseases. Recently, the expression of NLRP3 in CD4+ T cells, as well as in myeloid cells, has been described; however, a role of T cell–intrinsic NLRP3 in Treg differentiation remains unknown. Here, we report that NLRP3 impeded the expression of Foxp3 independent of inflammasome activation in Tregs. NLRP3-deficient mice elevate Treg generation in various organs in the de novo pathway. NLRP3 deficiency increased the amount and suppressive activity of Treg populations, whereas NLRP3 overexpression reduced Foxp3 expression and Treg abundance. Importantly, NLRP3 interacted with Kpna2 and translocated to the nucleus from the cytoplasm under Treg-polarizing conditions. Taken together, our results identify a novel role for NLRP3 as a new negative regulator of Treg differentiation, mediated via its interaction with Kpna2 for nuclear translocation.

Epidermal growth factor receptor (EGFR) supports colorectal cancer progression via oncogenic signaling. Anti-EGFR therapy is being investigated as a clinical option for colorectal cancer, and an observed interaction between EGFR and Prion protein has been detected in neuronal cells. We hypothesized that PrPC expression levels may regulate EGFR signaling and that detailed understanding of this signaling pathway may enable identification of resistance mechanisms and new actionable targets in colorectal cancer. We performed molecular pathway analysis following knockdown of PrPC or inhibition of EGFR signaling via gefitinib to identify changes in expression of key signaling proteins that determine cellular sensitivity or resistance to cisplatin. Expression of these proteins was examined in matched primary and metastatic patient samples and was correlated for resistance to therapy and progression of disease. Utilizing three colorectal cancer cell lines, we observed a correlation between high expression of PrPC and resistance to cisplatin. Investigation of molecular signaling in a resistant cell line revealed that PrPC contributed to signaling via colocalization with EGFR, which could be overcome by targeting p38 mitogen-activated protein kinases (p38 MAPK). We revealed that the level of Krüppel-like factor 5 (KLF5), a target downstream of p38 MAPK, was predictive for cell line and patient response to platinum agents. Further, high KLF5 expression was observed in BRAF-mutant colorectal cancer. Our study indicates that the EGFR to KLF5 pathway is predictive of patient progression on platinum-based therapy.

SB3 has been developed as a trastuzumab biosimilar, a therapeutic monoclonal antibody targeted to human epidermal growth factor receptor 2 (HER2), and approved by the European Commission and United States (US) Food and Drug Administration (FDA). During the developmental period of a biosimilar, setting an appropriate quality target is critical for assessing the similarity of the biosimilar product to the reference product. A stepwise approach should be taken to assessing similarity, beginning with extensive characterization of the reference product to establish the quality target. In this study, we evaluated the similarity of SB3 to the reference product and the impact of changes in the biological profile of the reference product on similarity assessment. Analytical similarity was assessed with defined test procedures in terms of critical quality attributes (CQAs) that could affect efficacy, potency, and safety, as well as for the non-CQAs that are related to process consistency. The quality target was established using up to 154 lots of European Union (EU)- and US-sourced Herceptin® (reference product), analyzed during the developmental period of SB3. Trends of the EU- and US-sourced reference product showed that the biological profile exhibited two marked changes for Fc-related attributes, and then recovered to pre-change quality level. Since the similarity range set by pre-change lots was considered most relevant, the changed lots were excluded from establishing the similarity range, which resulted in tightened acceptance criteria. As shown in the results of similarity assessment using the stringent quality target ranges, SB3 exhibits highly similar functional activities compared to the reference product in terms of both CQAs and non-CQAs. SB3 has been developed as a trastuzumab biosimilar approved in the EU and USA, and its manufacturing process is deemed to be robust and well-controlled within stringent quality target ranges.

Anthracyclines are amongst the most effective chemotherapeutics ever developed, but they produce grueling side-effects, serious adverse events and resistance often develops over time. We found that these compounds can be sequestered by secreted cellular Prion protein (PrPC), blocking their cytotoxic activity. This effect was dose-dependent using either cell line-conditioned medium or human serum as a source of PrPC. Genetic depletion of PrPC or inhibition of binding via chelation of ionic copper prevented the interaction and restored cytotoxic activity. This was more pronounced for doxorubicin than its epimer, epirubicin. Investigating the relevance to breast cancer management, we found that the levels of PRNP transcript in pre-treatment tumor biopsies stratified relapse-free survival after neoadjuvant treatment with anthracyclines, particularly amongst doxorubicin-treated patients with residual disease at surgery (p=2.8E-08). These data suggest that local sequestration could mediate treatment resistance. Consistent with this, tumor cell expression of PrPC protein correlated with poorer response to doxorubicin but not epirubicin in an independent cohort analyzed by immunohistochemistry, particularly soluble isoforms released into the extracellular environment by shedding (p=0.015). These findings have important potential clinical implications for frontline regimen decision-making. We suggest there is warranted utility for prognostic PrPC/PRNP assays to guide chemo-sensitization strategies that exploit an understanding of PrPC-anthracycline-copper ion complexes.

Hypoxia arises frequently in solid tumors and is a poor prognostic factor as it promotes tumor cell proliferation, invasion, angiogenesis, therapy resistance, and metastasis. Notably, there are two described forms of hypoxia present in a growing tumor: chronic hypoxia, caused by abnormal tumor vasculature, and intermittent hypoxia, caused by transient perfusion facilitated by tumor-supplying blood vessels. Here, we demonstrate that intermittent hypoxia, but not chronic hypoxia, endows breast cancer cells with greater metastatic potential. Using an immunocompetent and syngeneic murine model of breast cancer, we show that intermittent hypoxia enhances metastatic seeding and outgrowth in lungs in vivo. Furthermore, exposing mammary tumor cells to intermittent hypoxia promoted clonal diversity, upregulated metastasis-associated gene expression, induced a pro-tumorigenic secretory profile, increased stem-like cell marker expression, and gave rise to tumor-initiating cells at a relatively higher frequency. This work demonstrates that intermittent hypoxia, but not chronic hypoxia, induces a number of genetic, molecular, biochemical, and cellular changes that facilitate tumor cell survival, colonization, and the creation of a permissive microenvironment and thus enhances metastatic growth.

Transforming growth factor β (TGFβ) is overexpressed in advanced cancers and promotes tumorigenesis by inducing epithelial–mesenchymal transition (EMT), which enhances invasiveness and metastasis. Although we previously reported that EMT could be induced by increasing CK2 activity alone, it is not known whether CK2 also plays an essential role in TGFβ‐induced EMT. Therefore, in the present study, we investigated whether TGFβ signaling could activate CK2 and, if so, whether such activation is required for TGFβ‐induced EMT. We found that CK2 is activated by TGFβ treatment, and that activity peaks at 48 h after treatment. CK2 activation is dependent on TGFβ receptor (TGFBR) I kinase activity, but independent of SMAD4. Inhibition of CK2 activation through the use of either a CK2 inhibitor or shRNA against CSNK2A1 inhibited TGFβ‐induced EMT. TGFβ signaling decreased CK2β but did not affect CK2α protein levels, resulting in a quantitative imbalance between the catalytic α and regulatory β subunits, thereby increasing CK2 activity. The decrease in CK2β expression was dependent on TGFBRI kinase activity and the ubiquitin–proteasome pathway. The E3 ubiquitin ligases responsible for TGFβ‐induced CK2β degradation were found to be CHIP and WWP1. Okadaic acid (OA) pretreatment protected CK2β from TGFβ‐induced degradation, suggesting that dephosphorylation of CK2β by an OA‐sensitive phosphatase might be required for CK2 activation in TGFβ‐induced EMT. Collectively, our results suggest CK2 as a therapeutic target for the prevention of EMT and metastasis of cancers.

Tumor-derived exosomes are being recognized as essential mediators of intercellular communication between cancer and immune cells. It is well established that bone marrow-derived macrophages (BMDMs) take up tumor-derived exosomes. However, the functional impact of these exosomes on macrophage phenotypes is controversial and not well studied. Here, we show that breast cancer-derived exosomes alter the phenotype of macrophages through the interleukin-6 (IL-6) receptor beta (glycoprotein 130, gp130)-STAT3 signaling pathway. Addition of breast cancer-derived exosomes to macrophages results in the activation of the IL-6 response pathway, including phosphorylation of the key downstream transcription factor STAT3. Exosomal gp130, which is highly enriched in cancer exosomes, triggers the secretion of IL-6 from BMDMs. Moreover, the exposure of BMDMs to cancer-derived exosomes triggers changes from a conventional toward a polarized phenotype often observed in tumor-associated macrophages. All of these effects can be inhibited through the addition of a gp130 inhibitor to cancer-derived exosomes or by blocking BMDMs exosome uptake. Collectively, this work demonstrates that breast cancer-derived exosomes are capable of inducing IL-6 secretion and a pro-survival phenotype in macrophages, partially via gp130/STAT3 signaling.

A biosimilar product needs to demonstrate biosimilarity to the originator reference product, and the quality profile of the latter should be monitored throughout the period of the biosimilar's development to match the quality attributes of the 2 products that relate to efficacy and safety. For the development of a biosimilar version of trastuzumab, the reference product, Herceptin®, was extensively characterized for the main physicochemical and biologic properties by standard or state-of-the-art analytical methods, using multiple lots expiring between March 2015 and December 2019. For lots with expiry dates up to July 2018, a high degree of consistency was observed for all the tested properties. However, among the lots expiring in August 2018 or later, a downward drift was observed in %afucose (G0+G1+G2). Furthermore, the upward drift of %high mannose (M5+M6) was observed in the lots with expiry dates from June 2019 to December 2019. As a result, the combination of %afucose and %high mannose showed 2 marked drifts in the lots with expiry dates from August 2018 to December 2019, which was supported by the similar trend of biologic data, such as FcγRIIIa binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Considering that ADCC is one of the clinically relevant mechanisms of action for trastuzumab, the levels of %afucose and %high mannose should be tightly monitored as critical quality attributes for biosimilar development of trastuzumab.

Non-small cell lung cancer (NSCLC) is the most common lung cancer type and the most common cause of mortality in lung cancer patients. NSCLC is often associated with resistance to chemotherapeutics and together with rapid metastatic spread, results in limited treatment options and poor patient survival. NSCLCs are heterogeneous, and consist of epithelial and mesenchymal NSCLC cells. Mesenchymal NSCLC cells are thought to be responsible for the chemoresistance phenotype, but if and how this phenotype can be transferred to other NSCLC cells is currently not known. We hypothesised that small extracellular vesicles, exosomes, secreted by mesenchymal NSCLC cells could potentially transfer the chemoresistance phenotype to surrounding epithelial NSCLC cells. To explore this possibility, we used a unique human bronchial epithelial cell (HBEC) model in which the parental cells were transformed from an epithelial to mesenchymal phenotype by introducing oncogenic alterations common in NSCLC. We found that exosomes derived from the oncogenically transformed, mesenchymal HBECs could transfer chemoresistance to the parental, epithelial HBECs and increase ZEB1 mRNA, a master EMT transcription factor, in the recipient cells. Additionally, we demonstrate that exosomes from mesenchymal, but not epithelial HBECs contain the ZEB1 mRNA, thereby providing a potential mechanism for the induction of a mesenchymal phenotype in recipient cells. Together, this work demonstrates for the first time that exosomes derived from mesenchymal, oncogenically transformed lung cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells, likely via the transfer of ZEB1 mRNA in exosomes.What's new? In non-small cell lung cancer (NSCLC), chemoresistant phenotypes are enhanced among mesenchymal cells, while some other NSCLC cell subpopulations are inherently sensitive to chemotherapy. Whether the latter cells can receive a resistance phenotype via transfer from mesenchymal cells remains unclear. To identify a possible transfer mechanism, the authors of this study investigated small extracellular vesicles known as exosomes. In a human bronchial epithelial cell model, they show that mesenchymal NSCLC-derived exosomes from chemoresistant cells are capable of transferring resistance to chemosensitive epithelial cells. Recipient cells exhibited increased mRNA levels of the mesenchymal transcription factor ZEB1, suggesting a mechanism for chemoresistance induction.