Effect of exosome biomarkers for diagnosis and prognosis of breast cancer patients

Purpose

Exosomes are gradually detected as an indicator for diagnosis and prognosis of breast cancer in clinic and a systematic review was conducted.

Methods

A search for clinical studies published before July 1, 2017 was performed. Methods of exosome purification and identification from all studies were extracted. For diagnosis evaluation, the comparison of exosome biomarkers expression between breast cancer patients and healthy women was obtained; for prognosis prediction, the correlation between exosome biomarkers expression and chemotherapy resistance, overall survival (OS), disease-free survival (DFS), recurrence and metastasis of breast cancer was also extracted.

Results

A total of 11 studies with 921 breast cancer patients were included. Ultracentrifugation is the most frequent method to purify exosomes and transmission electron microscopy is commonly used to identify exosomes. Exosome biomarkers (such as HER2, CD47, Del-1, miR-1246 and miR-21) in breast cancer patients are significantly higher than those in healthy controls, exosomal GSTP1 and TRPC5 are related to chemotherapy resistance, exosome-carrying TRPC5, NANOG, NEUROD1, HTR7, KISS1R and HOXC are correlated to PFS, DFS or OS, and some exosomal proteins (HER2, KDR, CD49d, CXCR4 and CD44) as well as miRNAs (miR-340-5p, miR-17-5p, miR-130a-3p, miR-93-5p) are associated with tumor recurrence or distant organ metastasis.

Conclusions

Exosome biomarkers can be used for early diagnosis and prognosis of breast cancer patients in clinic.

     

Hepatocellular carcinoma-derived exosomal miRNA-21 contributes to tumor progression by converting hepatocyte stellate cells to cancer-associated fibroblasts

Background

Hepatocellular carcinoma (HCC) remains a global challenge due to its high morbidity and mortality rates as well as poor response to treatment. The communication between tumor-derived elements and stroma plays a critical role in facilitating cancer progression of HCC. Exosomes are small extracellular vesicles (EVs) that are released from the cells upon fusion of multivesicular bodies with the plasma membrane. There is emerging evidence indicating that exosomes play a central role in cell-to-cell communication. Much attention has been paid to exosomes since they are found to transport bioactive proteins, messenger RNA (mRNAs) and microRNA (miRNAs) that can be transferred in active form to adjacent cells or to distant organs. However, the mechanisms underlying such cancer progression remain largely unexplored.

Methods

Exosomes were isolated by differential ultracentrifugation from conditioned medium of HCC cells and identified by electron microscopy and Western blotting analysis. Hepatic stellate cells (HSCs) were treated with different concentrations of exosomes, and the activation of HSCs was analyzed by Western blotting analysis, wound healing, migration assay, Edu assay, CCK-8 assay and flow cytometry. Moreover, the different miRNA levels of exosomes were tested by real-time quantitative PCR (RT-PCR). The angiogenic ability of activated HSCs was analyzed by qRT-PCR, CCK-8 assay and tube formation assay. In addition, the abnormal lipid metabolism of activated HSCs was analyzed by Western blotting analysis and Oil Red staining. Finally, the relationship between serum exosomal miRNA-21 and prognosis of HCC patients was evaluated.

Results

We showed that HCC cells exhibited a great capacity to convert normal HSCs to cancer-associated fibroblasts (CAFs). Moreover, our data revealed that HCC cells secreted exosomal miRNA-21 that directly targeted PTEN, leading to activation of PDK1/AKT signaling in HSCs. Activated CAFs further promoted cancer progression by secreting angiogenic cytokines, including VEGF, MMP2, MMP9, bFGF and TGF-β. Clinical data indicated that high level of serum exosomal miRNA-21 was correlated with greater activation of CAFs and higher vessel density in HCC patients.

Conclusions

Intercellular crosstalk between tumor cells and HSCs was mediated by tumor-derived exosomes that controlled progression of HCC. Our findings provided potential targets for prevention and treatment of live cancer.

     

CD97 promotion of gastric carcinoma lymphatic metastasis is exosome dependent

Background

CD97 knockdown impairs the metastatic capacity of SGC-7901 gastric cancer cells. However, the role of CD97 in the distant lymphatic premetastatic niche formation of gastric cancer remains unknown.

Methods

Exosomes and the soluble fraction were isolated from SGC-L (an SGC-7901-cell-derived highly lymphatic metastatic cell line) and CD97-knockdown (SGC-L/CD97-kd) cells, and were co-cultured with gastric cancer cells. The metastatic capacity of the two cell lines was evaluated in vitro and in a footpad lymph node metastasis mouse model. Premetastatic-niche-formation-related proteins were examined immunohistochemically.

Results

CD97 expression was ninefold higher in SGC-L cells than in SGC-7901 cells. In vitro, exosomes or conditioned medium from the SGC-L cells enhanced cell proliferation (20 % increase) and invasion (30 % increase) as compared with that from SGC-L/CD97-kd cells (p < 0.01). Intrafootpad injections of SGC-L, but not SGC-L/CD97-kd exosomes or conditioned medium, strongly promoted SGC-L and SGC-L/CD97-kd cell accumulation in the draining lymph nodes (p < 0.01) and increased CD55, CD44v6, α₅β₁, CD31, epithelial cell adhesion molecule, and CD151 expression. Although the SGC-L/CD97-kd exosomes alone were insufficient for promotion of metastasis, they were partly aided by the SGC-L-cell-derived soluble fraction.

Conclusions

The CD97 small isoform promotes SGC-L cell lymphatic metastasis exosome dependently, and aided by the soluble fraction, the exosome-dependent CD97 plays a pivotal role in premetastatic niche formation.

     

MicroRNA-100 shuttled by mesenchymal stem cell-derived exosomes suppresses in vitro angiogenesis through modulating the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells

Background

Human mesenchymal stem cells (MSCs) have been shown to be involved in the formation and modulation of tumor stroma and in interacting with tumor cells, partly through their secretome. Exosomes are nano-sized intraluminal multi-vesicular bodies secreted by most types of cells and have been found to mediate intercellular communication through the transfer of genetic information via coding and non-coding RNAs to recipient cells. Since exosomes are considered as protective and enriched sources of shuttle microRNAs (miRNAs), we hypothesized that exosomal transfer of miRNAs from MSCs may affect tumor cell behavior, particularly angiogenesis.

Methods

Exosomes derived from MSCs were isolated and characterized by scanning electron microscopy analyses, dynamic light scattering measurements, and Western blotting. Fold changes in miR-100 expression levels were calculated in exosomes and their corresponding donor cells by qRT-PCR. The effects of exosomal transfer of miR-100 from MSCs were assessed by qRT-PCR and Western blotting of the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells. The quantification of secreted VEGF protein was determined by enzyme-linked immunosorbent assay. The putative paracrine effects of MSC-derived exosomes on tumor angiogenesis were explored by in vitro angiogenesis assays including endothelial cell proliferation, migration and tube formation assays.

Results

We found that MSC-derived exosomes induce a significant and dose-dependent decrease in the expression and secretion of vascular endothelial growth factor (VEGF) through modulating the mTOR/HIF-1α signaling axis in breast cancer-derived cells. We also found that miR-100 is enriched in MSC-derived exosomes and that its transfer to breast cancer-derived cells is associated with the down-regulation of VEGF in a time-dependent manner. The putative role of exosomal miR-100 transfer in regulating VEGF expression was substantiated by the ability of anti-miR-100 to rescue the inhibitory effects of MSC-derived exosomes on the expression of VEGF in breast cancer-derived cells. In addition, we found that down-regulation of VEGF mediated by MSC-derived exosomes can affect the vascular behavior of endothelial cells in vitro.

Conclusions

Overall, our findings suggest that exosomal transfer of miR-100 may be a novel mechanism underlying the paracrine effects of MSC-derived exosomes and may provide a means by which these vesicles can modulate vascular responses within the microenvironment of breast cancer cells.

     

Sortilin inhibition limits secretion-induced progranulin-dependent breast cancer progression and cancer stem cell expansion

Background

Cancer progression is influenced by genetic aberrations in the cancer cell population as well as by other factors including the microenvironment present within a tumour. Direct interactions between various cell types as well as cellular signalling via secreted cytokines can drive key tumourigenic properties associated with disease progression and treatment resistance. Also, cancer stem cell functions are influenced by the microenvironment. This challenging subset of cells has been linked to malignant properties. Within a screen, using in vivo like growth conditions, we identified progranulin as a highly secreted cytokine affecting cancer stem cells in breast cancer. This cytokine is known to play a role in numerous biological and tumour-related processes including therapy resistance in a range of cancer types.

Methods

Different in vitro and in vivo relevant conditions were used to validate breast cancer stem cell expansion mediated by progranulin and its receptor sortilin. Small interfering ribonucleic acid (siRNA) and pharmacological inhibition of sortilin were used to elucidate the role of sortilin as a functional receptor during progranulin-induced breast cancer stem cell propagation, both in vitro and in vivo, using breast cancer xenograft models. In addition, single-cell gene expression profiling as well as a Sox2 reporter breast cancer cell line were used to validate the role of dedifferentiation mediated by progranulin.

Results

In various in vivo-like screening assays, progranulin was identified as a potent cancer stem cell activator, highly secreted in ERα-negative breast cancer as well as in ERα-positive breast cancer under hypoxic adaptation. Progranulin exposure caused dedifferentiation as well as increased proliferation of the cancer stem cell pool, a process that was shown to be dependent on its receptor sortilin. Subcutaneous injections of progranulin or its active domain (GRN A) induced lung metastases in breast cancer xenograft models, supporting a major role for progranulin in cancer progression. Importantly, an orally bioavailable small molecule (AF38469) targeting sortilin, blocked GRN A-induced lung metastases and prevented cancer cell infiltration of the skin.

Conclusion

The collective results suggest that sortilin targeting represents a potential novel breast cancer therapy approach inhibiting tumour progression driven by secretion and microenvironmental influences.

     

Gastrokine 1 protein is a potential theragnostic target for gastric cancer

Background

Gastrokine 1 (GKN1) plays important roles in maintaining mucosal homeostasis, and in regulating cell proliferation and differentiation. Here, we determined whether GKN1 is a potential theragnostic marker for gastric cancer.

Methods

We identified GKN1 binding proteins using the protein microarray assay and investigated whether GKN1 is one of the exosomal cargo proteins by western blot, immunoprecipitation, and immunofluorescent assays. Cell proliferation and apoptosis were analyzed by MTT, BrdU incorporation, flow cytometry, and western blot assays. We further validated the functional relevance of exosomal GKN1 in MKN1-injected xenograft mice. The possibility of serum GKN1 as a diagnostic marker for gastric cancer was determined by ELISA assay.

Results

In protein microarray assay, GKN1 binding to 27 exosomal proteins was clearly observed. GKN1 was expressed in exosomes derived from HFE-145 gastric epithelial cells by western blot and immunofluorescent assays, but not in exosomes from AGS and MKN1 gastric cancer cells. Exosomes carrying GKN1 inhibited cell proliferation and induced apoptosis in both AGS and MKN1 cells, and exosomes carrying GKN1-treated nude mice-bearing MKN1 xenograft tumors exhibited significantly reduced tumor volume and tumor weight. Silencing of clathrin markedly down-regulated the internalization of exosomal GKN1. Interestingly, serum GKN1 concentrations in patients with gastric cancer were significantly lower than those in healthy individuals and patients with colorectal and hepatocellular carcinomas.

Conclusions

The GKN1 is secreted and internalized in the gastric epithelium by exosome-driven transfer, which inhibits gastric tumorigenesis and supports the clinical application of GKN1 protein in gastric cancer diagnosis and treatment.

     

Targeted therapies for treatment of renal cell carcinoma: recent advances and future perspectives

Purpose

A wide variety of targeted therapies are available for the treatment of renal cancer that has progressed beyond the point at which surgery is a viable option. In addition, there are many more that are in the different stages of clinical trials. Here, we provide a methodical discussion of the efficacy and safety of targeted therapies for the treatment of advanced renal cell carcinoma.

Methods

We conducted a systematic literature employing the search terms: renal cell carcinoma targets, tyrosine kinase inhibitors, mammalian target of rapamycin inhibitors, and each of the drugs discussed within these papers.

Results

The identified targeted therapies work by disrupting specific signalling pathways involved in tumour progression, such as those responsible for angiogenesis and cell proliferation. Tyrosine kinase inhibitors and mammalian target of rapamycin inhibitors are now established classes of drugs used in the treatment of renal cancer, with a total of six having received regulatory approval to date (sorafenib, sunitinib, pazopanib, axitinib, temsirolimus, and everolimus). Ongoing trials are likely to result in addition to these in the near future, for example, tivozanib, dovitinib, and cediranib. Furthermore, in addition to these small molecule drugs, immunotherapies involving monoclonal antibodies against signalling molecules such as vascular endothelial growth factor (bevacizumab) or programmed death-1 (nivolumab) are receiving increasing attention.

Conclusions

Targeted therapies have great potential for disrupting tumour progression by inhibiting certain signalling pathways. As our understanding of the biochemical pathways involved in cancer progresses, additional targets are certain to become apparent, expanding treatment options even further.

     

Anti-cell growth and anti-cancer stem cell activities of the non-canonical hedgehog inhibitor GANT61 in triple-negative breast cancer cells

Background

Triple-negative breast cancer (TNBC) exhibits biologically aggressive behavior and has a poor prognosis. Novel molecular targeting agents are needed to control TNBC. Recent studies revealed that the non-canonical hedgehog (Hh) signaling pathway plays important roles in the regulation of cancer stem cells (CSCs) in breast cancer. Therefore, the anti-cell growth and anti-CSC effects of the non-canonical Hh inhibitor GANT61 were investigated in TNBC cells.

Methods

The effects of GANT61 on cell growth, cell cycle progression, apoptosis, and the proportion of CSCs were investigated in three TNBC cell lines. Four ER-positive breast cancer cell lines were also used for comparisons. The expression levels of effector molecules in the Hh pathway: glioma-associated oncogene (GLI) 1 and GLI2, were measured. The combined effects of GANT61 and paclitaxel on anti-cell growth and anti-CSC activities were also investigated.

Results

Basal expression levels of GLI1 and GLI2 were significantly higher in TNBC cells than in ER-positive breast cancer cells. GANT61 dose-dependently decreased cell growth in association with G1–S cell cycle retardation and increased apoptosis. GANT61 significantly decreased the CSC proportion in all TNBC cell lines. Paclitaxel decreased cell growth, but not the CSC proportion. Combined treatments of GANT61 and paclitaxel more than additively enhanced anti-cell growth and/or anti-CSC activities.

Conclusions

The non-canonical Hh inhibitor GANT61 decreased not only cell growth, but also the CSC population in TNBC cells. GANT61 enhanced the anti-cell growth activity of paclitaxel in these cells. These results suggest for the first time that GANT61 has potential as a therapeutic agent in the treatment of patients with TNBC.

     

Pancreatic Cancer Metabolism: Molecular Mechanisms and Clinical Applications

Purpose of Review

Pancreatic adenocarcinoma is a leading cause of cancer mortality in western countries with a uniformly poor prognosis. Unfortunately, there has been little in the way of novel therapeutics for this malignancy over the last several decades. Derangements in metabolic circuitry favoring excess glycolysis are increasingly recognized as a key hallmark of cancer.

Recent Findings

The role of alterations in glutamine metabolism in pancreatic tumor progression has been elucidated in animal models and human cells lines, and there has been considerable interest in exploiting these aberrations for the treatment of pancreatic cancer. Other strategies targeting NQO1/GLS1 inhibition, NAD+ synthesis, and TCA cycle intermediates are being actively studied in the clinic.

Summary

Aberrant metabolism in pancreatic cancer poses a unique therapeutic strategy. We review preclinical and clinical studies looking to exploit alterations in the metabolic circuitry of pancreatic cancer.

     

Effects of hormone receptor status on the durable response of trastuzumab-based therapy in metastatic breast cancer

Purpose

We investigated whether miRNAs in exosomes from EMT6 or 4THM tumor-bearing mice played a role in regulating inflammatory cytokine expression and/or metastasis in WT mice injected with EMT6 and/or 4THM tumor cells.

Methods

EMT6 tumors in BALB/c CD200R1KO mice resolve following surgical resection of localized tumor and immunization with irradiated EMT6 cells along with CpG as adjuvant. Wild-type (WT) animals treated in the same fashion develop pulmonary and liver metastases within 20 days of surgery. DLNs from CD200R1KO mice contain no tumor cells detectable at limiting dilution. In contrast, 4THM tumor cells injected into CD200R1KO show increased metastasis compared with WT mice. Transfer of serum exosomes from 4THM tumor-bearing mice to WT animals increased metastasis of EMT6 tumors, an effect attenuated by anti-IL-6 antibody. We compared miRNA expression in exosomes from the serum of 4THM/EMT6 WT or CD200R1KO tumor-bearing mice, and the effects of antagomirs to miRNAs on tumor growth.

Results

Complex changes in miRNA expression were observed in the isolated exosomes. Some miRNAs, including miR155, have been reported to potentiate inflammatory responses and augment inflammatory cytokine expression. Expression of miR155 increased in exosomes from 4THM relative to EMT6 tumor bearers, and antagomirs to miR155 attenuated tumor growth and metastasis, and improved survival, following infusion into WT mice. Antagomirs to the miR205 family were thought to affect metastasis by targeting epithelial-to-mesenchymal transition (EMT), increased growth and metastasis in both 4THM and EMT6 tumor-bearing mice, and decreased survival, with some modulation of inflammatory cytokine production.

Conclusions

Multiple pathways are implicated in differential metastasis of EMT6/4THM, and targeting these may have clinical utility in human breast cancer.

     

Oncotype DX breast cancer recurrence score can be predicted with a novel nomogram using clinicopathologic data

Purpose

Breast cancer is a leading cause of cancer deaths in women, but despite steady improvements in therapies, treatment is still suboptimal. Immunotherapy holds promise as a more effective therapy for breast cancer; supporting this, our prior study showed that patients possessing HER2-reactive CD8+ T cells in blood experience survival superior to patients without these cells. Here, we define a composite set of biomarkers that identify patients with T cell responses to tumour antigens.

Methods

We assessed T cell responses following in vitro stimulation with the HER2, MUC1 and SUR tumour-associated antigens (TAA) by flow cytometry and intracellular cytokine staining in 50 breast cancer patients. We also measured HLA type, serum cytokines, tumour-infiltrating leukocytes and blood leukocyte populations.

Results

We found few correlations between TAA-reactive T cells and HLA type, serum cytokines and tumour-infiltrating leukocytes, whereas blood leukocyte phenotypes broadly correlated with TAA responses. This showed monocytes, natural killer cells, dendritic cells and T cells to be inversely associated with both CD4+ and CD8+ T cells reactive to tumour antigens. Moreover, combining multiple parameters improved the accuracy in predicting patients with TAA-responsive T cells.

Conclusion

This study therefore defines composite immune profiles that identify patients responding to TAAs which may allow better personalisation of cancer therapies.

     

Clinical outcomes linked to expression of gene subsets for protein hormones and their cognate receptors from LCM-procured breast carcinoma cells

Purpose

Certain peptide hormones and/or their cognate receptors influencing normal cellular pathways also have been detected in breast cancers. The hypothesis is that gene subsets of these regulatory molecules predict risk of breast carcinoma recurrence in patients with primary disease.

Methods

Gene expression levels of 61 hormones and 81 receptors were determined by microarray with LCM-procured carcinoma cells of 247 de-identified biopsies. Univariable and multivariable Cox regressions were determined using expression levels of each hormone/receptor gene, individually or as a pair.

Results

Molecular signatures for ER+/PR+, ER?/PR?, and ER? carcinoma cells deciphered by LASSO were externally validated at HRs (CI) of 2.8 (1.84–4.4), 1.53 (1.01–2.3), and 1.72 (1.15–2.56), respectively. Using LCM-procured breast carcinoma cells, a 16-gene molecular signature was derived for ER+/PR+ biopsies, whereas a 10-gene signature was deciphered for ER?/PR? cancers. Four genes, POMC, CALCR, AVPR1A, and GH1, of this 10-gene signature were identified in a 6-gene molecular signature for ER? specimens.

Conclusions

Applying these signatures, Kaplan–Meier plots definitively identified a cohort of patients with either ER?/PR? or ER? carcinomas that exhibited low risk of recurrence. In contrast, the ER+/PR+ signature identified a cohort of patients with high risk of breast cancer recurrence. Each of the three molecular signatures predicted clinical outcomes of breast cancer patients with greater accuracy than observed with either single-gene analysis or by ER/PR protein content alone. Collectively, our results suggest that gene expression profiles of breast carcinomas with suspected poor prognosis (ER?/PR?) have identified a subset of patients with decreased risk of recurrence.

     

Cyclin-dependent kinase 4/6 inhibitors in breast cancer: palbociclib,ribociclib, and abemaciclib

Purpose

There is a growing body of literature demonstrating that immune-related expression signatures predict breast cancer prognosis and chemo-/targeted-therapy responsiveness. However, it is unclear whether these signatures correlate with each other or represent distinct immune-related signals.

Methods

We evaluated 57 published immune-related expression signatures in four public breast cancer datasets totaling 3295 samples. For each dataset, we used consensus clustering to group signatures together based on their co-expression pattern. Signatures that were in the same consensus cluster across all four datasets were used to define immune modules. Tumors were then classified into immune subtypes based on their module scores using consensus clustering. Survival analysis was conducted using Cox proportional hazards modeling.

Results

Consensus clustering consistently yields four distinct co-expression modules across the datasets. These modules appear to represent distinct immune components and signals, where constituent signatures relate to (1) T-cells and/or B-cells (T/B-cell), (2) interferon (IFN), (3) transforming growth factor beta (TGFB), and (4) core–serum response, dendritic cells, and/or macrophages (CSR). Subtyping of tumors based on these co-expression modules consistently yields subsets that fall into five major immune subtypes: T/B-cell/IFN High, IFN/CSR High, CSR High, TGFB High, and Immune Low. Basal and/or triple-negative breast cancer patients with CSR High tumors have significantly worse outcome relative to those within the T/B-cell/IFN High subtype.

Conclusion

Our exploratory study identified four distinct immune co-expression modules (T/B-cell, IFN, TGFB, or CSR) from published immune signatures. Using these modules, we identified five immune subtypes with significant outcome differences in basal breast cancers.