Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

Lung cancer is the leading cause of cancer-related deaths in the world, and non-small cell lung cancer (NSCLC) is the most common subset. We previously found that infiltration of tumor inflammatory monocytes (TIMs) into lung squamous carcinoma (LUSC) tumors is associated with increased metastases and poor survival. To further understand how TIMs promote metastases, we compared RNA-Seq profiles of TIMs from several LUSC metastatic models with inflammatory monocytes (IMs) of non-tumor-bearing controls. We identified Spon1 as upregulated in TIMs and found that Spon1 expression in LUSC tumors corresponded with poor survival and enrichment of collagen extracellular matrix signatures. We observed SPON1+ TIMs mediate their effects directly through LRP8 on NSCLC cells, which resulted in TGF-ß1 activation and robust production of fibrillar collagens. Using several orthogonal approaches, we demonstrated that SPON1+ TIMs were sufficient to promote NSCLC metastases. Additionally, we found that Spon1 loss in the host, or Lrp8 loss in cancer cells, resulted in a significant decrease of both high-density collagen matrices and metastases. Finally, we confirmed the relevance of the SPON1/LRP8/TGF-ß1 axis with collagen production and survival in patients with NSCLC. Taken together, our study describes how SPON1+ TIMs promote collagen remodeling and NSCLC metastases through an LRP8/TGF-ß1 signaling axis.

Horizontal transfer of miR-383 sensitise cells to cisplatin by targeting VEGFA-Akt signalling loop.

BACKGROUND: Cellular resistance to cisplatin has been one of the major obstacles in the success of combination therapy for many types of cancers. Emerging evidences suggest that exosomes released by drug resistant tumour cells play significant role in conferring resistance to drug sensitive cells by means of horizontal transfer of genetic materials such as miRNAs. Though exosomal miRNAs have been reported to confer drug resistance, the exact underlying mechanisms are still unclear. METHODS AND RESULTS: In the present study, mature miRNAs secreted differentially by cisplatin resistant and cisplatin sensitive HepG2 cells were profiled and the effect of most significantly lowered miRNA in conferring cisplatin resistance when horizontally transferred, was analysed. we report miR-383 to be present at the lowest levels among the differentially abundant miRNAs expressed in exosomes secreted by cisplatin resistant cells compared to that that of cisplatin sensitive cells. We therefore, checked the effect of ectopic expression of miR-383 in altering cisplatin sensitivity of Hela cells. Drug sensitivity assay and apoptotic assays revealed that miR-383 could sensitise cells to cisplatin by targeting VEGF and its downstream Akt mediated pathway. CONCLUSION: Results presented here provide evidence for the important role of miR-383 in regulating cisplatin sensitivity by modulating VEGF signalling loop upon horizontal transfer across different cell types.

Integrin ß4 induced epithelial-to-mesenchymal transition involves miR-383 mediated regulation of GATA6 levels.

BACKGROUND: The process of transdifferentiating epithelial cells to mesenchymal-like cells (EMT) involves cells gradually taking on an invasive and migratory phenotype. Many cell adhesion molecules are crucial for the management of EMT, integrin ß4 (ITGB4) being one among them. Although signaling downstream of ITGB4 has been reported to cause changes in the expression of several miRNAs, little is known about the role of such miRNAs in the process of EMT. METHODS AND RESULTS: The cytoplasmic domain of ITGB4 (ITGB4CD) was ectopically expressed in HeLa cells to induce ITGB4 signaling, and expression analysis of mesenchymal markers indicated the induction of EMT. ß-catenin and AKT signaling pathways were found to be activated downstream of ITGB4 signaling, as evidenced by the TOPFlash assay and the levels of phosphorylated AKT, respectively. Based on in silico and qRT-PCR analysis, miR-383 was selected for functional validation studies. miR-383 and Sponge were ectopically expressed in HeLa, thereafter, western blot and qRT-PCR analysis revealed that miR-383 regulates GATA binding protein 6 (GATA6) post-transcriptionally. The ectopic expression of shRNA targeting GATA6 caused the reversal of EMT and ß catenin activation downstream of ITGB4 signaling. Cell migration assays revealed significantly high cell migration upon ectopic expression ITGB4CD, which was reversed upon ectopic co-expression of miR-383 or GATA6 shRNA. Besides, ITGB4CD promoted EMT in in ovo xenograft model, which was reversed by ectopic expression of miR-383 or GATA6 shRNA. CONCLUSION: The induction of EMT downstream of ITGB4 involves a signaling axis encompassing AKT/miR-383/GATA6/ß-catenin.

Periostin+ Stromal Cells Guide Lymphovascular Invasion by Cancer Cells.

Cancer cell dissemination to sentinel lymph nodes is associated with poor patient outcomes, particularly in breast cancer. The process by which cancer cells egress from the primary tumor upon interfacing with the lymphatic vasculature is complex and driven by dynamic interactions between cancer cells and stromal cells, including cancer-associated fibroblasts (CAF). The matricellular protein periostin can distinguish CAF subtypes in breast cancer and is associated with increased desmoplasia and disease recurrence in patients. However, as periostin is secreted, periostin-expressing CAFs are difficult to characterize in situ, limiting our understanding of their specific contribution to cancer progression. Here, we used in vivo genetic labeling and ablation to lineage trace periostin+ cells and characterize their functions during tumor growth and metastasis. Periostin-expressing CAFs were spatially found at periductal and perivascular margins, were enriched at lymphatic vessel peripheries, and were differentially activated by highly metastatic cancer cells versus poorly metastatic counterparts. Surprisingly, genetically depleting periostin+ CAFs slightly accelerated primary tumor growth but impaired intratumoral collagen organization and inhibited lymphatic, but not lung, metastases. Periostin ablation in CAFs impaired their ability to deposit aligned collagen matrices and inhibited cancer cell invasion through collagen and across lymphatic endothelial cell monolayers. Thus, highly metastatic cancer cells mobilize periostin-expressing CAFs in the primary tumor site that promote collagen remodeling and collective cell invasion within lymphatic vessels and ultimately to sentinel lymph nodes. SIGNIFICANCE: Highly metastatic breast cancer cells activate a population of periostin-expressing CAFs that remodel the extracellular matrix to promote escape of cancer cells into lymphatic vessels and drive colonization of proximal lymph nodes.

Regulation of poly ADP-ribosylation of VEGF by an interplay between PARP-16 and TNKS-2.

The biological activity of vascular endothelial growth factor (VEGF), the major cytokine regulating the process of angiogenesis is tightly controlled at multiple levels including processes involving post-translational modification such as ADP-ribosylation and glycosylation. ADP-ribosylation is a reversible NAD+-dependent modification, catalyzed by poly ADP-ribose polymerase (PARP) or ADP-ribosyl transferase (ADPRTs) and has been reported by us and others as a modification that reduces the biological activity of VEGF. The factors responsible for any such modification should occur in the secretory pathway, i.e., in the endoplasmic reticulum and Golgi. Our investigation carried out in this direction revealed that ADP-ribosylation of VEGF requires the interplay between members of poly ADP-ribose polymerase (PARP) family in the secretory pathway, viz., ER associated PARP-16 and Golgi associated Tankyrase-2 (TNKS-2). The data presented in this manuscript suggest that PARP-16 catalysis the priming mono ADP-ribosylation of VEGF which is a prerequisite for poly ADP-ribosylation of VEGF by TNKS-2.

Role of Sirtuins in Tumor Angiogenesis.

Generally, changes in the metabolic status of cells under conditions like hypoxia and accumulation of lactate can be sensed by various sensing mechanisms, leading to modulation of a number of signal transduction pathways and transcription factors. Several of the proangiogenic cytokines like VEGF, FGF, PDGF, TGF-ß, Ang-2, ILs, etc. are secreted by cancer cells, under hypoxic microenvironment. These cytokines bind to their receptors on the endothelial cells and activates a number of signaling pathways including Akt/PIP3, Src, p38/MAPK, Smad2/3, etc., which ultimately results in the proliferation and migration of endothelial cells. Transcription factors that are activated in response to the metabolic status of tumors include HIFs, NF-κb, p53, El-2, and FOXO. Many of these transcription factors has been reported to be regulated by a class of histone deacetylase called sirtuins. Sirtuins are NAD+ dependent histone deacetylases that play pivotal role in the regulation of tumor cell metabolism, proliferation, migration and angiogenesis. The major function of sirtuins include, deacetylation of histones as well as some non-histone proteins like NF-κB, FOXOs, PPAR⋎, PGC1-α, enzymes like acetyl coenzymeA and structural proteins like α tubulin. In the cell, sirtuins are generally considered as the redox sensors and their activities are dependent on the metabolic status of the cell. Understanding the intricate regulatory mechanisms adopted by sirtuins, is crucial in devising effective therapeutic strategies against angiogenesis, metastasis and tumor progression. Keeping this in mind, the present review focuses on the role of sirtuins in the process of tumor angiogenesis and the regulatory mechanisms employed by them.

ER stress mediated regulation of miR23a confer Hela cells better adaptability to utilize glycolytic pathway.

Cancer cells exhibit increased dependency on aerobic glycolysis, a phenomenon referred as the "Warburg effect" and therefore, blocking glycolysis by using non-metabolizable analogues of glucose, like 2-Deoxy glucose (2-DG), has been proposed to be of huge therapeutic importance. One of the major drawbacks of using 2-DG as a chemotherapeutic agent is that it can induce ER stress. ER stress is a hall mark in many solid tumors and the unfolded protein response (UPR) associated with it initiates many survival mechanisms in cancer cells. In the present study, we report a novel survival mechanism associated with ER stress, by which the cancer cells become more adapted to aerobic glycolysis. When ER stress was induced in Hela cells by treating them with 2-DG or Thapsigargin (TG) the expression and activity of LDH was significantly up regulated, conferring the cells a greater glycolytic potential. A simultaneous decrease was observed in the expression of miR-23a, which was predicted in silico to have target site on the 3'UTR of LDH A and B mRNAs. miRNA over expression studies and mRNA degradation assays suggest that miR-23a could target LDH A and LDH B mRNAs. Further on the basis of our results and previous scientific reports, we propose that "c-Myc," which is over expressed during ER stress, repress the expression of miR-23a, which in turn regulates the expression of its target genes viz., LDH A and LDH B, thereby making the cells more competent to survive in tumor microenvironment, which requires efficient use of aerobic glycolysis.

Horizontal transfer of miR-23a from hypoxic tumor cell colonies can induce angiogenesis.

Neo vessel formation by angiogenesis is an important event during many pathological conditions including cancer, where it is indispensable for tumor growth and survival. Although, various pro-angiogenic cytokines and soluble factors, secreted by tumor cells, have been reported to promote angiogenesis, recent studies have shown regulatory role of exosomes, secreted by tumor cells in the process of angiogenesis. These exosomes are capable of carrying nucleic acids, proteins, etc., as their cargo. Under the light of these facts and considering the presence of miRNAs, the non-coding RNAs capable of regulating target gene expression, as one of the major cargos in the exosomes, we investigated, whether exosomes derived from normoxic and hypoxic tumor cell colonies exhibit difference in levels of miR-23∼27∼24 cluster members and if so, to check the significance of their horizontal transfer on the process of angiogenesis. Results of our study showed that exosomes secreted by hypoxic tumor cell colonies possess significantly higher levels of miR23a and can induce angiogenesis. Further, we have shown that exosomes secreted by cells that ectopically over express miR23a is capable of inducing angiogenesis in different angiogenic model systems such as CAM, in ovo Xenograft and HUVEC models systems. Further, mechanistic analysis revealed that miR23a driven regulation of angiogenesis is brought about by down regulation of SIRT1 in the recipient cells. Collectively, the results presented here suggest that exosomal transfer of miR23a from tumor cell colonies can induce the process of angiogenesis by targeting SIRT1 in the recipient endothelial cells.

MicroRNA106a regulates matrix metalloprotease 9 in a sirtuin-1 dependent mechanism.

Cellular migration is important during many physiological as well as pathological conditions and is regulated very tightly by an intricate network of signaling and effector molecules. One of the important players during cellular migration are matrix metalloproteases and their levels have been reported to be important in determining the cellular migratory properties during metastasis. MMPs and regulators of MMPs therefore, present themselves as potent candidates for manipulation, to control conditions where they get dysregulated. Micro RNAs are a group of micro regulators that can modulate expression of a gene through transcriptional and post transcriptional regulations. Owing to the fact that many microRNAs have already been reported to regulate MMPs and that miR106a, a member of oncomir17 family has been implicated in metastatic conditions, the present study intended to analyze if miR106a can regulate levels of MMP9, an important inducible matrix metalloproteinase. The results of the in vitro experiments demonstrated that under conditions of migration cells showed elevated levels of miR106a, which could regulate the expression of major MMP9 regulator, SIRT-1. Decreased levels of SIRT1thus resulted in an increase in the expression and activity of MMP9. Over expression and mRNA stability studies carried out also suggested regulatory role of miR106a. The overall results thus suggested that the levels of miR106a gets modulated during cellular migration, causing a change in the levels of SIRT-1 mRNA by affecting its stability and the levels of SIRT-1 in turn can regulate the levels of MMP9.

Horizontal transfer of miR-106a/b from cisplatin resistant hepatocarcinoma cells can alter the sensitivity of cervical cancer cells to cisplatin.

Recent studies indicate that horizontal transfer of genetic material can act as a communication tool between heterogenous populations of tumour cells, thus altering the chemosensitivity of tumour cells. The present study was designed to check whether the horizontal transfer of miRNAs released by cisplatin resistant (Cp-r) Hepatocarcinoma cells can alter the sensitivity of cervical cancer cells. For this exosomes secreted by cisplatin resistant and cisplatin sensitive HepG2 cells (EXres and EXsen) were isolated and characterised. Cytotoxicity analysis showed that EXres can make Hela cells resistant to cisplatin. Analysis of miR-106a/b levels in EXres and EXsen showed that their levels vary. Mechanistic studies showed that miR-106a/b play an important role in EXsen and EXres mediated change in chemosensitivity of Hela cells to cisplatin. Further SIRT1 was identified as a major target of miR-106a/b using in silico tools and this was proved by experimentation. Also the effect of miR-106a/b in chemosensitivity was seen to be dependent on regulation of SIRT1 by miR-106a/b. In brief, this study brings into light, the SIRT1 dependent mechanism of miR-106a/b mediated regulation of chemosensitivity upon the horizontal transfer from one cell type to another.

2-Deoxy Glucose Modulates Expression and Biological Activity of VEGF in a SIRT-1 Dependent Mechanism.

Reprogramming of energy metabolism particularly switching over of cells to aerobic glycolysis leading to accumulation of lactate is a hallmark of cancer. Lactate can induce angiogenesis, an important process underlying tumor growth and metastasis. VEGF is one of the most important cytokines which regulate this process and the present study was designed to examine if blocking glycolytic pathway in tumor cells can affect its angiogenic potency with respect to VEGF. For this, the expression and biological activity of VEGF synthesized and secreted by tumor derived cell lines in the presence or absence of 2-deoxy glucose (2-DG), an inhibitor of glycolysis was determined. The results suggested that inhibition of glycolysis using sub-lethal doses of 2-DG down-regulated the expression of VEGF and also significantly reduced its biological activity. Further mechanistic studies revealed that the down regulation of VEGF gene expression by 2-DG was due to an increase in SIRT-1 activity and the reduced biological activity was found to be due to an increase in the PAR modification of VEGF. Activity of SIRT-1 and PAR modification of VEGF in turn, was found to be correlated to the cellular NAD+ levels. The results presented here therefore suggest that treatment of cancer cells with 2-DG can significantly reduce its overall angiogenic potency through transcriptional and post-translational mechanisms. J. Cell. Biochem. 118: 252-262, 2017. © 2016 Wiley Periodicals, Inc.

2-Deoxy glucose regulate MMP-9 in a SIRT-1 dependent and NFkB independent mechanism.

MMP9 is a member of the family of zinc-containing endopeptidases which degrade various components of the extracellular matrix, thereby regulating matrix remodeling. Since matrix remodeling plays an important role during growth and progression of cancer and considering the fact that, tumor cells switch to aerobic glycolysis as its major energy source, this study was designed to analyze if partial inhibition of glycolysis (the major energy pathway during hypoxia) can be used as a means to control matrix remodeling in terms of MMP9 activity and expression. For this, human epithelial carcinoma cells were treated with glycolytic inhibitor, 2-deoxy glucose (2DG) at sub-lethal concentrations followed by analysis of the expression and activity of MMP2 and MMP9. The experimental findings demonstrate that exposure of cancer cells to glycolytic inhibitor at concentration that does not induce ER stress, downregulates the activity and expression of MMP9 without affecting the expression levels and activity of MMP2. Further mechanistic analysis revealed that the regulation of MMP9 was mediated in a SIRT-1 dependent mechanism and did not alter the NFkB signaling pathway. The overall results presented here, therefore suggest that the use of glycolytic inhibitor, 2DG at concentration that do not affect cell viability or induce ER stress can be an effective strategy to control matrix remodeling.