The antidiabetic drug metformin exerts chemopreventive and antineoplastic effects in many types of malignancies. However, the mechanisms responsible for metformin actions appear diverse and may differ in different types of cancer. Understanding the molecular and cellular mechanisms specific for different cancers is important to optimize strategy for metformin treatment in different cancer types. Here, we investigate the in vitro and in vivo effects of metformin on esophageal squamous cell carcinoma (ESCC) cells. Metformin selectively inhibited cell growth in ESCC tumor cells but not immortalized noncancerous esophageal epithelial cells. In addition to apoptosis, metformin triggered autophagy. Pharmacological or genetic inhibition of autophagy sensitized ESCC cells to metformin-induced apoptotic cell death. Mechanistically, signal transducer and activator of transcription 3 (Stat3) and its downstream target Bcl-2 was inactivated by metformin treatment. Accordingly, small interfering RNA (siRNA)-mediated Stat3 knockdown enhanced metformin-induced autophagy and apoptosis, and concomitantly enhanced the inhibitory effect of metformin on cell viability. Similarly, the Bcl-2 proto-oncogene, an inhibitor of both apoptosis and autophagy, was repressed by metformin. Ectopic expression of Bcl-2 protected cells from metformin-mediated autophagy and apoptosis. In vivo, metformin downregulated Stat3 activity and Bcl-2 expression, induced apoptosis and autophagy, and inhibited tumor growth. Together, inactivation of Stat3-Bcl-2 pathway contributes to metformin-induced growth inhibition of ESCC by facilitating crosstalk between apoptosis and autophagy.

Drug resistance is a daunting challenge in the treatment of breast cancer (BC). Exosomes, as intercellular communicative vectors in the tumor microenvironment, play an important role in BC progression. With the in-depth understanding of tumor heterogeneity, an emerging role of exosomes in drug resistance has attracted extensive attention. The functional proteins or non-coding RNAs contained in exosomes secreted from tumor and stromal cells mediate drug resistance by regulating drug efflux and metabolism, pro-survival signaling, epithelial–mesenchymal transition, stem-like property, and tumor microenvironmental remodeling. In this review, we summarize the underlying associations between exosomes and drug resistance of BC and discuss the unique biogenesis of exosomes, the change of exosome cargo, and the pattern of release by BC cells in response to drug treatment. Moreover, we propose exosome as a candidate biomarker in predicting and monitoring the therapeutic drug response of BC and as a potential target or carrier to reverse the drug resistance of BC.

Background: The tRNA-derived small RNAs (tsRNAs) are produced in a nuclease-dependent manner in responses to variety of stresses that are common in cancers. We focus on a cancer-enriched tsRNA signature to develop a salivary exosome-based non-invasive biomarker for human esophageal squamous cell carcinoma (ESCC). Methods: Cancer-enriched small RNAs were identified by RNA sequencing of salivary exosomes obtained from ESCC patients (n = 3) and healthy controls (n = 3) in a pilot study and further validated in discovery cohort (n = 66). A multicenter prospective observational study was conducted in two ESCC high-incidence regions (n = 320 and 200, respectively) using the newly developed biomarker signature. Results: The tsRNA (tRNA-GlyGCC-5) and a previously undocumented small RNA were specifically enriched in salivary exosomes of ESCC patients, ESCC tissues and ESCC cells. The bi-signature composed of these small RNAs was able to discriminate ESCC patients from the controls with high sensitivity (90.50%) and specificity (94.20%). Based on the bi-signature Risk Score for Prognosis (RSP), patients with high-RSP have both shorter overall survival (OS) (HR 4.95, 95%CI 2.90–8.46) and progression-free survival (PFS) (HR 3.69, 95%CI 2.24–6.10) than those with low-RSP. In addition, adjuvant therapy improved OS (HR 0.47, 95%CI 0.29–0.77) and PFS (HR 0.36, 95%CI 0.21–0.62) only for patients with high but not low RSP. These findings are consistent in both training and validation cohort. Conclusions: The tsRNA-based signature not only has the potential for diagnosis and prognosis but also may serve as a pre-operative biomarker to select patients who would benefit from adjuvant therapy. Trial registration: A prospective study of diagnosis biomarkers of esophageal squamous cell carcinoma, ChiCTR2000031507. Registered 3 April 2016 - Retrospectively registered.

Purpose: The tumor immune microenvironment (TIME) has an important impact on response to cancer immunotherapy using immune checkpoint inhibitors. Specifically, an “infiltrated-excluded”/“cold” TIME is predictive of poor response. The antidiabetic agent metformin may influence anticancer immunity in esophageal squamous cell carcinoma (ESCC). Experimental Design: We analyzed matched pre- and posttreatment ESCC specimens in a phase II clinical trial of low-dose metformin treatment (250 mg/day) to evaluate direct anti-ESCC activity and TIME reprogramming. Follow-up correlative studies using a carcinogen-induced ESCC mouse model were performed with short-term (1 week) or long-term (12 weeks) low-dose metformin (50 mg/kg/day) treatment. Results: In the clinical trial, low-dose metformin did not affect proliferation or apoptosis in ESCC tumors as assayed by Ki67 and cleaved caspase-3 immunostaining. However, metformin reprogrammed the TIME toward “infiltrated-inflamed” and increased the numbers of infiltrated CD8+ cytotoxic T lymphocyte and CD20+ B lymphocyte. Further, an increase in tumor-suppressive (CD11c+) and a decrease in tumor-promoting (CD163+) macrophages were observed. Metformin augmented macrophage-mediated phagocytosis of ESCC cells in vitro. In the ESCC mouse model, short-term metformin treatment reprogrammed the TIME in a similar fashion to humans, whereas long-term treatment further shifted the TIME toward an active state (e.g., reduction in CD4+ FoxP3+ regulatory T cells) and inhibited ESCC growth. In both humans and mice, metformin triggered AMPK activation and STAT3 inactivation, and altered the production of effector cytokines (i.e., TNFα, IFNγ, and IL10) in the immune cells. Conclusions: Low-dose metformin reprograms the TIME to an activated status and may be a suitable immune response modifier for further investigation in patients with ESCC.

The current biomarkers available in the clinic are not enough for early diagnosis or for monitoring disease progression of ovarian cancer. Liquid biopsy is a minimally invasive test and has the advantage of early diagnosis and real-time monitoring of treatment response. Although significant progress has been made in the usage of circulating tumor cells and cell-free DNA for ovarian cancer diagnosis, their potential for early detection or monitoring progression remains elusive. Extracellular vesicles (EVs) are a heterogeneous group of lipid membranous particles released from almost all cell types. EVs contain proteins, mRNA, DNA fragments, non-coding RNAs, and lipids and play a critical role in intercellular communication. Emerging evidence suggests that EVs have crucial roles in cancer development and metastasis, thus holding promise for liquid biopsy-based biomarker discovery for ovarian cancer diagnosis. In this review, we discuss the advantages of EV-based liquid biopsy, summarize the protein biomarkers identified from EVs in ovarian cancer, and highlight the utility of new technologies recently developed for EV detection with an emphasis on their use for diagnosing ovarian cancer, monitoring cancer progression, and developing personalized medicine.