microRNAs as mediators and communicators between cancer cells and the tumor micro-environment

Abstract

Cancer cells grow in an environment comprised of multiple components that support tumor growth and contribute to therapy resistance. Major cell types in the tumor micro-environment are fibroblasts, endothelial cells and infiltrating immune cells all of which communicate with cancer cells. One way that these cell types promote cancer progression is by altering expression of miRNAs, small noncoding RNAs that negatively regulate protein expression, either in the cancer cells or in associated normal cells. Changes in miRNA expression can be brought about by direct interaction between the stromal cells and cancer cells, by paracrine factors secreted by any of the cell types, or even through direct communication between cells through secreted miRNAs. Understanding the role of miRNAs in the complex interactions between the tumor and cells in its micro-environment is necessary if we are to understand tumor progression and devise new treatments.

Introduction

The tumor micro-environment

The tumor micro-environment (TME) is composed of fibroblasts, blood vessels, immune cells, support cells, signaling molecules, and the extracellular matrix (ECM). The proportion of this stroma in human cancers can be over 90% ^{1, 2}. The tumor and the surrounding micro-environment affect each other through close and constant interactions and together play a significant role in treatment outcomes ³ (Figure 1).

Figure 1.

An overview of the role of miRNAs in the tumor microenvironment.

Endothelial cells line the interior surface of blood vessels and lymphatic vessels, forming an interface between circulating blood or lymph in the lumen and the rest of the vessel wall. In tumors, aggressive and unregulated growth of neoplastically transformed cells that overexpress pro-angiogenic factors leads to the development of disorganized blood vessel networks that are fundamentally different from normal vasculature ³.

Cancer associated fibroblasts (CAFs) are a major constituent of the tumor stroma ^{4, 5}. CAFs isolated from cancer patients have a different morphology and function than normal fibroblasts and have been shown to promote the invasion and growth of tumor cells ⁵. CAFs produce growth factors (VEGF) and cytokines (TGFβ, Interleukin(IL)-6, IL-10) that activate the adjacent ECM, which then contributes to the growth of the cancer cells. Additionally, CAFs are the primary source of an altered ECM, containing fibronectin and collagen, that also contributes to tumor growth ⁴. Major secreted factors include proinflammatory cytokines such as IL-1β and IL-8 which have been associated with pro-tumorigenic effects. A prominent chemokine that is secreted by CAFs is SDF-1 α, which primarily signals through the CXC chemokine receptor 4 (CXCR4) which promotes proliferation ⁴. CAFs retain their characteristics (at least partially) when cultured in the absence of malignant cells, suggesting that they have undergone genetic or epigenetic alterations. A recent series of studies reported genetic alterations in microdissected CAFs derived from breast, head and neck, and ovarian cancer ^4-6. However, careful analysis of breast and ovarian CAFs demonstrated that such genetic alterations are extremely rare ⁷. It is more likely that the stable phenotype of CAFs is regulated by epigenetic changes, as suggested by a genome-wide analysis of breast cancer stroma ⁸.

Many cancers are driven by inflammation mediated by bone-marrow derived cells, such as monocytes and macrophages. Macrophages are innate immune cells that play indispensable roles in the innate and adaptive immune response. They can be activated by a variety of stimuli and polarized to functionally different phenotypes. Two distinct subsets of macrophages are now recognized: classically activated (M1) and alternatively activated (M2) macrophages ⁹. M1 macrophages express a series of proinflammatory cytokines, chemokines, and effector molecules, such as TNF- α, IL-12, IL-23, iNOS and MHC class I and II. In contrast, M2 macrophages express a wide array of anti-inflammatory molecules, such as IL-10, TGF- β, and arginase. In most tumors, infiltrating macrophages are of the M2 phenotype, which provides an immunosuppressive micro-environment for tumor growth. In addition, these tumor-associated macrophages secrete many cytokines, chemokines, and proteases, which also promote tumor angiogenesis, growth, metastasis, and immunosuppression ⁹. This review is about the role that microRNAs play in the communication between tumor cells and components of the TME (Figure 1).

miRNAs and cancer

Micro (mi)RNAs are small, 19-22 nucleotide (nt) long, non-coding RNAs that inhibit gene expression at the posttranscriptional level. They are first transcribed as parts of longer molecules, up to several kilobases in length (pri-miRNA), that are processed in the nucleus into hairpin RNAs of 70-100 nt by the double-stranded RNA-specific ribonuclease, Drosha ¹⁰. The hairpin pre-miRNAs are then transported by exportin 5 to the cytoplasm, where they undergo further processing by a second, double-strand specific ribonuclease, called Dicer. In animals, single-stranded miRNAs are incorporated into RNA induced silencing complex (RISC) and primarily bind specific messenger RNA (mRNA) at specific sequence motifs, predominantly within the 3′ untranslated region (3′UTR) of the transcript, that are significantly, though not completely, complementary to the miRNA. The mRNA/miRNA duplex then inhibits translation by blocking initiation or through increased degradation of the mRNA. Given the frequency with which miRNA target motifs are conserved within 3′UTRs, it is estimated that almost all human genes are conserved targets of miRNAs, and that for each miRNA there are approximately 200 genes that carry conserved sequence motifs within the 3′UTR ¹⁰.

miRNAs, which are frequently deregulated in many types of cancer, have been shown to play roles in multiple facets of tumorigenesis. The cancer cell autonomous roles of miRNAs have been thoroughly reviewed elsewhere ¹¹. In this review the focus will be on the non-cell autonomous roles of miRNAs in cancer cells, specifically in how alterations in miRNA expression in cancer cells promotes tumorigenesis by modifying the TME. The modification of the TME through the regulation of miRNA expression by oncogenes was first reported in 2006 ¹². Using a colonocyte model with stable expression of Ras or Ras and myc, Dews et al. demonstrated that myc upregulates the miR-17-92 family, therefore promoting blood perfusion and endothelial cell recruitment ¹².

A strong relationship between miRNAs and human cancers has been established. A comparison of miRNA expression in normal and tumor tissues showed global changes in miRNA expression in various human malignancies ¹⁰. In addition, mapping 186 human miRNA genes revealed that they are frequently located at fragile sites and other cancer-associated chromosomal regions ¹³. Consequently, miRNAs have been identified either as oncogenes (e.g. miR-155, miR-17-5p, miR-21, miR-221/222) or tumor suppressors (e.g. miR-34, miR-15a, miR-16-1, let-7) ^{10, 14}. Thus, while miRNA deregulation in cancer cells plays a known role in tumor progression, metastasis, and therapy resistance, miRNAs have recently been shown to participate in reprogramming components of the TME to provide assistance for the cancer organ.

Reprogramming of somatic cells by miRNAs

Until 9 years ago, it was believed that most differentiated tissues were irreversibly locked into their differentiation states and that tissues could only be generated with pluripotent stem cells. This changed in 2006, with the demonstration that the expression of 4 transcription factors could reprogram somatic fibroblasts into induced pluripotent stem (iPS) cells ¹⁵. Since then, miRNAs were identified that are expressed only in embryonic stem cells and it was reported that miR-302 ¹⁶ could reprogram somatic cells into iPS cells ¹⁷. In addition, it was demonstrated that expression of two miRNAs (miR-9/9* and miR-124) could convert fibroblasts into neurons ¹⁸, proving the power of miRNAs to reprogram adult fibroblasts. These data showed that the genome has much more plasticity than previously expected and that miRNAs are powerful regulators of this plasticity. In the context of cancer, the idea developed that investigating the reprogramming of cancer associated stroma cells could lead to a better understanding of cancer progression and to new treatments. This review highlights a number of miRNAs and their roles in reprogramming major components of the TME and discuss miRNAs that change in the cancer cells in response to interaction with components of the TME.

The role of miRNAs in cancer associated endothelial cells (CAEs)

Since endothelial cells are critical for angiogenesis and regulating tumor metastasis, their recruitment by cancer cells can substantially promote overall tumorigenesis. Through manipulation of the miRNA expression of endothelial cells, the cancer cells are able to reprogram these cells to enhance their angiogenic potential (Figure 2).

Figure 2.

Deregulated miRNAs in cancer associated endothelial cells (CAE) or cancer cells interacting with CAE. When known miRNA targets are given. BrCa: breast cancer; HCC, hepatocellular carcinoma; OvCa: ovarian cancer; CRC: colorectal cancer. References: [1], ²¹; [2], ¹⁹; [3], ²⁰; [4], ²²; [5], ²³; [6], ²⁵; [7], ²⁴; [8], ³⁶; [9], ¹²; [10], ²⁹; [11], ²⁷; [12], ³⁰; [13], ³¹; [14], ³⁷; [15], ³²; [16], ³³; [17], ³⁹; [18], ³⁴; [19], ³⁵.

In two different cancer models, a similar approach was taken to determine how changes in miRNA expression in endothelial cells affect co-cultured cancer cells. When endothelial cells were co-cultured with hepatocellular carcinoma (HCC) cells, 4 miRNAs were significantly altered (>1.5 fold) in the endothelial cells, with miR-146a, miR-181a*, and miR-140-5p upregulated and miR-302c downregulated ¹⁹. The upregulation of miR-146a was found to promote endothelial cell migration and proliferation and to promote tumor growth and vascularization ¹⁹. This activity occurred through miR-146a directly targeting BRCA-1, which then negatively regulated expression of PDGFRA through targeting the PDGFRA promoter ¹⁹. When endothelial cells were co-cultured with glioma cell lines, a miRNA array analysis identified 12 miRNAs that were downregulated (miR-181a, miR-101, miR-30b, miR-27a, miR-21, miR-22, miR-23b, miR-31, miR-103, miR-126, miR-29a, and miR-125b) and one upregulated (miR-296) ²⁰. miR-296 was found to promote angiogenesis through increasing endothelial tube formation and migration. The glioma cells reprogrammed the endothelial cells to upregulate miR-296 through VEGF and EGF ²⁰. It was also shown that VEGF upregulates expression of miR-10b and miR-196b in murine breast and lung cancer models ²¹. These two miRNAs were found to be critical for angiogenesis and tumor growth in these cancers ²¹.

miR-125b is another miRNA that clearly has an important, though at present difficult to define, role in endothelial cell reprogramming during tumorigenesis. In gliomas a miRNA array detected the downregulation of miR-125b, which was confirmed to have a role in endothelial migration ^{22, 23}. Once again VEGF played a role in reprogramming the endothelial cells through altering miRNA expression. VEGF downregulated miR-125b, leading to the upregulation of MAZ (Myc-associated zinc finger protein), which is a transcription factor that binds the promoter of VEGF. This process in the endothelial cells clearly promoted vascularization and angiogenesis ²³. However, in another report miR-125b was shown to be upregulated by VEGF, FGF, and hypoxia in murine lung cancer models resulting in reduced vascularization and tumorigenesis ²². Whether these contradictions concerning the role of miR-125b in the reprogramming of endothelial cells are based on differences in cancer model or on experimental reasons needs to be further explored.

In breast cancer, alterations in miRNA expression have been shown to regulate angiogenesis through direct effects on endothelial cells and immune infiltration. Specifically, the upregulation of miR-155 and miR-93 ^{24, 25} or the downregulation of miR-126 and miR-29b ^{26, 27} have been linked to angiogenesis through effects on endothelial cells, while downregulation of miR-92a may play a role in macrophage infiltration ²⁸. miR-126 was shown to directly target both IGFBP2, which is a secreted endothelial recruitment factor and PITPNC1, the phosphatidylinositol transfer protein that regulates IGFBP2 secretion. Thus, the downregulation of miR-126 enhances endothelial recruitment and tumor growth by increasing IGFBP2 secretion. miR-126 was also shown to employ another distinct mechanism for modifying the chemotactic gradient through targeting MERTK, which when secreted binds to and neutralizes the negative chemotactic factor, GAS6. Therefore, the downregulation of miR-126 resulted in the upregulation of MERTK, which, by neutralizing physiological levels of GAS6, led to enhanced endothelial cell chemotaxis ²⁷. Indeed, miR-126 downregulation was found to be necessary for endothelial cell recruitment in this breast cancer model ²⁷. The upregulation of miR-155 has also been shown to be important in breast cancer, by promoting angiogenesis through the enhanced migration of endothelial cells and macrophages ²⁵. miR-155 directly targets VHL, which is critical for the proangiogenic effects of the increased expression of miR-155 ²⁵. Additionally, miR-93 was found to be upregulated in breast carcinoma patients ²⁴. Increased expression of miR-93 enhanced endothelial tube formation, increased blood vessel density in vivo, and promoted the formation of lung metastases through targeting of LATS2 ²⁴. The altered expression of miR-29b, which was downregulated through the loss of GATA3, also promoted tumorigenesis through enhanced angiogenesis, resulting in a poor prognosis in luminal breast cancer. Downregulation of miR-29b enhanced TGF-β signaling and promoted an overall epithelial-to-mesenchymal transition (EMT) ²⁹. Furthermore, expression of VEGFA, ITGA6, ANGPTL4, and LOX, known mediators of angiogenesis, were inversely correlated with expression of miR-29b ²⁹. Moreover, miRNA changes in cancer cells, as well as endothelial cells are important in breast cancer. The downregulation of miR-92a in the cancer cells correlated with a worse prognosis and reduced macrophage infiltration ²⁸.

miR-29b, miR-214, and miR-195 are also downregulated in hepatocellular carcinoma (HCC) and coordinate the TME through the regulation of endothelial cells. As is the case in breast cancer, downregulation of miR-29b in HCC enhanced tumorigenesis through increased microvessel density and endothelial tube formation ²⁶. This effect may be due to an increase in MMP-2 expression caused by the downregulation of miR-29b, which promotes VEGFR (also known as Flt-1) signaling in the endothelial cells ²⁶. Another miRNA, miR-195, directly targets VEGF in HCC ³⁰. The downregulation of miR-195 increased expression of VEGF, leading to enhanced endothelial cell branching and tube formation during co-culture ³⁰. An analysis of a miRNA array of tumors from HCC patients, found that miR-214 was highly downregulated and that this downregulation was indicative of a worse prognosis ³¹. miR-214 was found to regulate endothelial cell branching through targeting HDGF, which is known to be involved with angiogenesis ³¹.

In lung cancer, downregulation of miR-519c, miR-126, and let-7b was found to promote angiogenesis through effects on endothelial cells. Additionally, downregulation of miR-200b was shown to promote metastasis through the regulation of CAFs and the regulation of endothelial cell recruitment in renal and ovarian cancer as well as lung cancer. miR-519c, which is downregulated by HGF, regulated angiogenesis by targeting HIF-1α ³². The downregulation of miR-519c in lung cancer promoted the secretion of proangiogenic factors, VEGF-A, bFGF, and IL-8, which enhanced endothelial tube formation and increased blood vessel density within the TME ³². Additionally, it was shown that downregulation of miR-126 and let-7b in both the tumor and the stroma was associated with increased microvessel density and a worse prognosis ³³. While the mechanism and mediators of this increased microvessel density are not known, this observation suggests a role of miRNAs in angiogenesis in lung cancer.

Downregulation of miR-200 family members has been found to affect endothelial cell activity, and has been shown, through analysis of the TGCA dataset, to indicate a worse prognosis in lung, renal, and ovarian cancer ³⁴. While the role of miR-200 in EMT is well known, it was shown that the downregulation of miR-200 family members reduced the amount of endothelial cells in the TME through targeting IL-8 ³⁴. Furthermore, increased expression of miR-200 in endothelial cells reduced migration and tube formation indicating that miR-200 is a regulator of angiogenesis in the TME ³⁴.

Evidence of epigenetic modification of miRNA expression to promote angiogenesis was seen in ovarian cancer, melanoma, and glioblastoma. In addition to the downregulation of miR-200 in ovarian cancer, miR-484 was found to be downregulated in ovarian cancer patient tumor samples and was linked to an increase in blood vessels, possibly through targeting VEGFB and VEGFR2 ³⁵. In melanoma, in vivo passaging of cancer cells to generate highly metastatic clones, indicated that miR-1908, miR-214, miR-199a-5p, and miR-199-3p were upregulated and functional mediators of metastasis ³⁶. Increased expression of miR-1908, miR-199a-5p, and miR-199a-3p indicated a worse prognosis ³⁶. These miRNAs were shown to regulate microvessel density, endothelial cell recruitment, and melanoma metastasis and their increased expression was linked to a worse prognosis. Their mechanism of action was through targeting DNAJA4 and also ApoE, which reduces endothelial cell recruitment by binding the LRP8 receptor on endothelial cells ³⁶. A similar approach, using in vivo passaging, was taken with glioblastoma. A human glioblastoma line was passaged in nude mice to identify clones with different dormancy rates determined by rate of outgrowth upon challenge ³⁷. In the fast growing glioblastoma lines, miR-580, miR-588, and miR-190 were downregulated, which increased proangiogenic factors TGFα, HIF-1α, FGF2, TIMP-3, and Kras ³⁷. Reconstitution of miR-588 in fast growing glioblastoma cell lines reduced recruitment of endothelial cells and MDSCs ³⁷. Furthermore, increased expression of miR-93 in glioblastoma promoted endothelial cell proliferation and migration, possibly through targeting integrin B8 ³⁸.

In summary, many of the epigenetic changes in miRNA expression in cancer involve the recruitment of endothelial cells, which promote angiogenesis. However, a large number of studies identified other cells within the TME that change miRNA expression in the setting of cancer.

The role of miRNAs in cancer associated fibroblasts (CAFs)

Fibroblasts regulate angiogenesis, metastasis, EMT and extracellular matrix composition within the tumor. Not surprisingly, miRNAs are key regulators of the tumor promoting functions of CAFs and it is becoming more readily apparent that cancer directed changes regulate this miRNA network. In a large number of cancers CAFs have been shown to promote tumorigenesis by upregulating and downregulating specific miRNAs (Figure 3).

Figure 3.

Deregulated miRNAs in cancer associated fibroblasts (CAF) or cancer cells interacting with CAF. When known miRNA targets are given. BrCa: breast cancer; OvCa: ovarian cancer; CRC: colorectal cancer; Panc: pancreatic cancer; Gastr: gastric cancer; PrCa: prostate cancer; Eso squam: esophageal squamous cell carcinoma. References: [1], ⁴⁹; [2], ⁴⁸; [3], ⁴⁰; [4], ⁴¹; [5], ⁴²; [6], ⁴³; [7], ⁴⁶]; [8], ⁴⁴; [9], ⁵²; [10], ⁵³; [11], ⁵¹; [12], ⁵⁰; [13], ¹¹⁷.

During tumorigenesis miRNAs are frequently downregulated in what has been considered a dedifferentiation mechanism. More specifically, lung cancer cells downregulate miR-200b, which targets Flt-1 to promote CAF migration that increase metastasis ³⁹. While the majority of miRNAs that are altered in the micro-environment are downregulated, the increase of several miRNAs of note has been shown to promote tumorigenesis. These include well known oncomirs such as miR-21 and miR-155. miR-21 has been shown to be highly upregulated in fibroblasts from patients in multiple tumor types, including colorectal cancer ^40-42, squamous cell carcinoma ⁴³, and pancreatic cancer ⁴⁴. Increased expression of miR-21 by fibroblasts was shown to upregulate α -SMA, resulting in the acquisition of a more activated myofibroblast phenotype ^{40, 45}. Cancer cells regulated miR-21 through soluble factors, including TGF- β, which has been shown to be relevant in several different cancer models ^{40, 44, 45}. For example, TGF- β was shown to upregulate miR-143 in CAFs in scirrhous type gastric cancer ⁴⁶. These results highlight that regulation of tumorigenesis by “oncomirs” is not cancer autonomous, but rather a function of the entire tumor organ.

As previously mentioned, miR-155 is often upregulated in CAFs. miR-155 has a prominent role in inflammation, since it is directly regulated by NF- κ B ⁴⁷, a well-known regulator of cancer associated fibroblast function. We recently reported that upregulation of miR-155 contributed to the transition of normal omental fibroblasts into CAFs in ovarian cancer ⁴⁸. Gene expression analysis of these CAFs and the effects of the upregulation of miR-155 showed an increase in the expression of chemokines and other immune modulators which may be congruent with the effects of miR-155 and its known regulator, NF- κ B. However further dissection of the role of miR-155 and its overlap with NF- κ B in the promotion of tumor growth by CAFs will need to be pursued further, since this study focused on the coordinated deregulation of 3 miRNAs (the upregulation of miR-155 with the downregulation of both miR-31 and miR-214) ⁴⁸.

A miRNA array was also used to identify altered signaling pathways in fibroblasts from breast cancer patients. miR-221-5p, miR-221-3p and miR-31-3p were found to be upregulated in these CAFs ⁴⁹. The authors then performed gene ontology analysis, searching for pathways that were regulated by multiple deregulated miRNAs. Many pathways were identified, including MAPK, IL-6, tight junction, insulin, chemokine, TGF- β, and HGFR signaling ⁴⁹. Additional analysis validated the activation of the IL-6 pathway and downstream signaling of TGF- β with increased MMP-1, MMP-2, and MMP-9 in CAFs from breast cancer ⁴⁹. However, the role and significance of these upregulated miRNAs in regulating CAFs will need to be further dissected because miR-205, miR-200c, miR-200b, miR-141, miR-101, miR-342-3p, let-7g, and miR-26b were also shown to be significantly downregulated ⁴⁹.

Interestingly, the study discussed above, which performed a miRNA array to identify deregulated expression in of CAFs in breast cancer found that miR-31 was upregulated ⁴⁹ while the report on ovarian cancer metastases found that miR-31 was downregulated. ⁴⁸. Further profiling of miRNA expression in endometrial cancer showed that miR-31 is downregulated in fibroblasts ⁵⁰. This downregulation of miR-31 permitted the expression of SATB2, promoting endometrial cancer invasiveness and growth (49). The apparent paradox of the different regulation of miR-31 in different types of cancer may reflect the distinct tissue origin that determines the unique context for the growth of each individual tumor. Interestingly, in endometrial cancer the downregulation of miR-148a, as well as miR-31, in CAFs is critical to the promotion of tumorigenesis. The downregulation of miR-148 in CAFs increases Wnt activity through elevated expression of Wnt10B, which is a target of miR-148 ⁵¹.

Another study that performed miRNA array analysis on CAFs in breast cancer reported 6 miRNAs that were downregulated more then 10-fold, including miR-7f, let-7g, miR-107, miR-15b, miR-26b, and miR-30b ⁵². The downregulation of miR-26b in CAFs promoted the migration of fibroblasts, which is a functional determinant of the CAF phenotype ⁵². Furthermore, the downregulation of miR-26b in CAFs promoted the invasion of the human breast cancer cell line, MCF-7 ⁵². Additionally, PTEN expression was reduced in the stroma of invasive breast cancer patients, which was correlated with miR-320 expression ^{53, 54}. Loss of PTEN expression in stromal fibroblasts led to a protumorigenic secretome, which was partially mediated by the downregulation of miR-320. Furthermore, the authors determined that miR-320 directly regulated the transcription factor ETS2, and ETS2 binding sites were found in 20 genes of the 54 gene secretome identified through PTEN deletion ⁵³. The downregulation of miR-320 in fibroblasts promoted cancer cell invasion and endothelial proliferation through direct targeting of MMP-9 and EMILIN2, respectively. EMILIN2 is a secreted factor important for angiogenesis through the promotion of endothelial cell proliferation ⁵⁵. That miR-320 regulated expression of MMP-9 and EMILIN2 both directly and indirectly was indicated by the presence of ETS2 binding sites that are responsible for their regulation. Thus, miR-320 targets the upstream transcription factor and the downstream mediators regulating the protumorigenic secretome of fibroblasts ⁵³.

In prostate cancer, as in breast cancer, miRNAs were downregulated in CAFs to promote tumorigenesis. In prostate cancer the downregulation of miR-15 and miR-16 in CAFs was likely mediated through activation of the AKT and ERK pathways, promoting prostate cancer migration and neovascularization (55). This data adds to our awareness of the diversity of miRNA regulation in fibroblasts, based on different tumor types and other unknown physiological factors.

The role of miRNAs in cancer associated mesothelial cells

While ovarian cancer interacts with CAFs, the primary micro-environment for ovarian cancer cells is the mesothelial cell. These cells cover the peritoneum, the small and large bowel serosa and the omentum. The most common sites of metastasis from the primary ovarian tumor are the abdominal peritoneum and the omentum. Metastasis is mediated by binding of ovarian cancer cells to mesothelial cells before they can access to the fibroblasts located underneath. Recently, it was shown that TGF β -stimulated human primary mesothelial cells, which induced expression of MMP-2 and MMP-9, were able to promote cancer cell attachment and proliferation through downregulating members of the miR-200 family. Consequently, the delivery of miR-200 family miRNAs to mesothelial cells in mice inhibited ovarian cancer cell implantation and dissemination ⁵⁶. We recently showed that the direct interaction of OvCa cells with mesothelial cells, which cover the surface of the omentum, causes a DNA methyltransferase 1 (DNMT1) mediated decrease in the expression of miR-193b in the cancer cells ⁵⁷. The reduction in miR-193b resulted in the upregulation of its target urokinase-type plasminogen activator (uPA), a known tumor associated protease. The paracrine signals emitted from the TME resulted in the deregulation of a key miRNA in the cancer cells, promoting the initial steps of OvCa metastatic colonization.

The deregulation of miRNAs in tumor-associated macrophages promotes tumorigenesis

Cancer cells reprogram and regulate the miRNA expression of infiltrating immune cells, not only to suppress an anti-tumor immune response but to highjack the wound healing response of these cells to promote tumorigenesis. Antigen presenting cells, especially macrophages, have been shown to be a primary target of this conversion. They represent an ideal target, given their juxtaposition between the innate and adaptive immune system. Macrophages that infiltrate the TME adopt an M2 polarized phenotype, which promotes tumor growth more than the inflammatory M1 phenotype. As expected, cancer cells upregulate and downregulate several miRNAs in immune cells to limit their anti-tumor response and reprogram them to tumorigenesis (Figure 4).

Figure 4.

Deregulated miRNAs in cancer associated macrophages (CAM) or cancer cells interacting with CAM. When known miRNA targets are given. BrCa: breast cancer; HCC, hepatocellular carcinoma; OvCa: ovarian cancer; CRC: colorectal cancer; Panc: pancreatic cancer; Gastr: gastric cancer. References: [1], ⁷⁰; [2], ⁶⁹; [3], ⁶⁷; [4], ⁹²; [5], ⁶⁰; [6], ⁶²; [7], ⁶⁴; [8], ⁷¹; [9], ⁸⁷; [10], ⁸⁸; [11], ⁷²; [12], ⁵⁸; [13], ⁶⁸; [14], ⁶³.

miR-155 is a well-known regulator of the immune response, involving T cells, NK cells, B cells, and antigen presenting cells (macrophages and dendritic cells). Notably, miR-155 has been found to be persistently downregulated in tumor associated macrophages (TAMs) ^{58, 59}. Soluble factors from HCC were shown to downregulate miR-155, increasing expression of the transcription factor C/EPB β and increasing production of IL-10 ⁵⁸. When expression of miR-155 was restored in macrophages or dendritic cells, both direct and indirect anti-tumor responses were promoted through the enhancement of T cell function. miR-155 targeted SOCS1, C/EPB β and possibly Ship1, which are transcriptional suppressors of Th1 type cytokine production (e.g. TNF α, IL-12, IFN γ, CCL5) ^{58, 60, 61}. Several other miRNAs were also found to be downregulated within TAMs, including miR-142-3p ⁶², miR-125b ⁶³, and miR-19a-3p ⁶⁴. Downregulation of miR-142-3p limited the tumor infiltration of macrophages and reduced the therapeutic effect of adoptive T cell transfer ⁶². Restoring miR-125b expression in macrophages promoted an anti-tumor response by targeting IRF-4, a known promotor of the M2 macrophage phenotype ^{63, 65}. Macrophages have been found to downregulate miR-19a-3p within the TME, possibly through a cancer derived soluble factor ⁶⁴. The downregulation of miR-19a-3p promoted STAT3 signaling, because miR-19a-3p directly targets Fra-1 (Fos-related antigen), a member of the AP1 family involved in macrophage polarization and the upstream regulation of STAT3 ^{64, 66}. STAT3 expression in macrophages, which promotes immune suppression, was rescued upon overexpression of miR-19a-3p ⁶⁴. In contrast, in pancreatic cancer miR-146a is upregulated in monocytes to prevent their differentiation and expression of costimulatory molecules ⁶⁷. This is a similar mechanism of immune evasion as is seen with the downregulation of miR-142-3p, which is not only critical for macrophage infiltration but also for macrophage differentiation ⁶². Additionally, elevated levels of miR-200 in myeloid cells reduced angiogenesis through the targeting of VEGF that limited tumor growth in a murine lung cancer model ⁶⁸.

miR-511-3p is part of an interesting negative feedback loop in TAMs. in which it is coordinately regulated with the mannose receptor, a marker for TAMs. However, instead of promoting tumorigenesis, miR-511-3p negatively regulates protumoral gene expression ⁶⁹. This indicates that not all miRNA reprogramming in macrophages will drive tumorigenesis, but can be the result of negative feedback limiting the protumorigenic capabilities of TAMs.

The role of miRNAs in myeloid derived suppressor cells

Myeloid derived suppressor cells (MDSCs) are components of the myeloid compartment that, like TAMs, regulate immunosuppression within the TME. Therefore, these MDSCs can, through cancer directed miRNA reprogramming, suppress immune responses and promote tumor growth. A miRNA array identified 8 deregulated miRNAs in tumor infiltrating MDSCs in a murine breast cancer model. miR-494, miR-882, and miR-361 were upregulated and let-7e, miR-713, miR-133b, miR-466j, and miR-322 downregulated ⁷⁰. miR-494 was the most significantly upregulated miRNA, and promoted breast cancer invasion through suppression of anti-tumor CD8 T cell responses, possibly in response to TGF- β, ⁷⁰. miR-494 was shown to target PTEN in MDSCs, which is responsible for the enhanced immune suppression of CD8 T cells ⁷⁰. Further evidence suggests that several miRNAs are downregulated in MDSCs to promote the differentiation of myeloid cells into MDSCs and to regulate immunosuppressive signaling pathways. The downregulation of miR-223 in the bone marrow were critical if myeloid progenitors were to differentiate into MDSCs in a murine breast cancer model. Stable overexpression of miR-223 prevented the differentiation into MDSCs and the ability to promote tumor growth through immune suppression ⁷¹. miR-17-5p and miR-20a regulated the immunosuppressive potential of MDSCs through targeting of STAT3 ⁷². Cancers downregulate these miRNAs, increasing the expression of STAT3, which in turn enhanced immunosuppression, partially through STAT3 induction of reactive oxygen species production⁷².

It is evident, therefore, that cancer cells reprogram the myeloid compartment to evade the immune system and promote tumorigenesis. These miRNA alterations directly limit immune responses through polarizing macrophages from a M1 to a M2 phenotype and indirectly through reprogramming macrophages and MDSCs to inhibit adaptive immune responses. However, there is now growing evidence that immune cells within the TME are, themselves, able to regulate miRNA expression in cancer cells. Soluble factors secreted from macrophages have been shown to downregulate miR-7 in gastric cancer and promote tumorigenesis ⁷³. Additionally, co-culture experiments pairing astrocytes with three different tumor types that commonly metastasize to the brain (melanoma, breast, and lung) showed that astrocytes downregulate miR-768-3p when exposed to any cancer cells ⁷⁴. In addition, the downregulation of miR-768-3p was found to promote chemo resistance and cancer stem cell properties ⁷⁴. In a model of ovarian cancer, MDSCs were shown to upregulate miR-101 and promote cancer “stemness” and miR-101 was shown to target CtBP2, which bound to the promoters of Sox2 and Nanog ⁷⁵. Follicular dendritic cells promoted chemoresistance in follicular lymphoma and mantle cell lymphoma through upregulation of miR-181a ⁷⁶. Follicular dendritic cells are commonly found adhered to lymphoma cells. This interaction was shown to upregulate miR-181a, which targets the BH3 only protein Bim, making the lymphoma cells less sensitive to mitoxantrone ⁷⁶.

The role of miRNAs in tumor infiltrating T cells (TILs)

Components of the adaptive immune system paradoxically play two roles in cancer with CD8 T cells and CD4 T cells performing tumor surveillance while T regulatory cells (Tregs) suppress anti-tumor responses. To avoid immune detection and elimination cancer cells directly suppress T cells through multiple mechanisms including deregulating miRNA expression. Further, cancer cells indirectly subvert the immune system through enhancing the suppressive functions of adaptive immune cells like Tregs and NKT cells through altering miRNA expression in these cells. In addition to regulating miRNAs in TILs, cancer cells have been shown to alter their own miRNA programming in order to avoid immune detection.

CD4 T cells isolated from patients with glioblastoma multiforme (GBM) show repressed expression of miR-17∼92 family members, while the miR-17∼92 family was shown to be generally upregulated in Th1 type CD4 T cells ⁷⁷. This suggests a skewing of T cells towards a Th2 phenotype in the TME. Additionally, TGF- β, an immune suppressive cytokine commonly elevated in cancer, suppresses anti-tumor T cell responses by upregulating miR-23a in TILs, which targets BLIMP-1 reducing CTL expression of anti-tumor effector molecules, granzyme B and IFN γ ⁷⁸. As previously discussed miR-155, is a miRNA associated with inflammation in the TME. Increased expression of miR-155 in T cells was shown to promote anti-tumor responses ^{77, 79}. miR-155 expression in T cells negatively correlated with SOCS1 and SHIP1, regulators of T cell activation and function ^{78, 79}. Furthermore, miR-155 in conjunction with miR-146a in T cells regulates IFN γ production ⁷⁹. miRNAs are not only deregulated in effector T cells. A miRNA array comparison between peripheral Tregs and tumor infiltrating Tregs demonstrated deregulation of several miRNAs which may be regulated by Foxp3 ⁸⁰. These studies demonstrate that cancer cells regulate miRNAs in T cells that are important for anti-tumor T cell responses.

Avoidance of immune surveillance has recently been included as a hallmark of cancer. Cancer cells alter expression of transcription factors, surface receptors, soluble chemokines/cytokines and now it appears miRNAs to subvert the immune system. In GBM miR-124 was significantly downregulated compared to normal brain tissue. Downregulation of miR-124 increased Treg infiltration and reduced TIL cytokine production potentially through altered expression of STAT-3, a target of miR-124 ⁸¹. Further in portal vein tumor thrombosis, TGF- β was shown to downregulate miR-34a, which targets the Treg recruitment chemokine CCL-22 ⁸². Increasing expression of CCL-22 promoted Treg infiltration ⁸². Thus, demonstrating that deregulation of miRNAs within the cancer cells can alter the TME through manipulation of Tregs. In addition to Tregs, NKT cells may suppress the TILs as well ⁸³. Glioma cells were shown to increase the proportion of IL-10 and IL-6 producing NK cells possibly through the upregulation of miR-92a, which suppressed NKT responsiveness and possibly CD8 T cell function ⁸⁴. HCC cell lines were shown to alter expression of miR-222 and miR-339 to target ICAM-1 reducing anti-tumor T cell responses ⁸⁵. In addition to cancer cells altering miRNA expression to avoid the immune system, cancer cells may regulate miRNA expression to use the immune system to promote metastasis. T cells that infiltrated prostate cancer produced FGF11, which upregulated miR-541 in prostate cancer cells. The upregulation of miR-541 enhanced in vitro invasion of prostate cancer potentially through MMP-9 and androgen receptor signaling ⁸⁶. Collectively these data support the notion that the widespread changes in miRNA expression in cancer cells promote tumorigenesis and determine the behavior of cancer including, immune evasion.

miRNAs, MSCs, and cancer cells

Mesenchymal stem cells (MSCs) are routinely recruited in solid tumors in order to support tumorigenesis through enhancing angiogenesis, metastasis, and immune modulation. Given that miRNAs regulate protein expression, influencing the differentiation and activation of cells, it is not surprising that cancer cells direct changes in miRNA expression in MSCs to reprogram them into tumor promoting cells. MSCs from breast adipose tissue were found to downregulate miR-15b, miR-27b, and miR-29b in response to conditioned media from human breast cancer cell lines. The downregulation of these miRNAs, which promoted MSC migration, was induced by soluble factors ⁸⁷. Interestingly, miRNA arrays of breast cancer cell lines with differing degrees of metastatic potential indicated that miR-126/126* were significantly downregulated in the more metastatic cell lines ⁸⁸. Since these miRNAs target SDF-1, a chemokine known to recruit MSCs to the TME, their downregulation promoted breast cancer metastasis by increasing the recruitment of MSCs ^88-90. Collectively, these studies demonstrate that, in breast cancer, miRNA changes in both the MSCs and cancer cells foster enhanced recruitment of MSCs to the cancer. Furthermore, MSCs downregulate miR-200b and miR-200c in MCF-7 cells through TGF- β, resulting in the upregulation of ZEB1 and ZEB2, suggesting a role for MSCs in promoting EMT ⁹¹. Thus, miRNA reprogramming in both MSCs and breast cancer cells is involved in both the recruitment of MSCs to the TME and the protumorigenic effects of MSCs in the TME. Moreover, the ability of MSCs to reprogram cancer cells is not limited to breast cancer. In HCC, MSCs upregulated miR-155 through secretion of S100A4, a calcium binding protein that has a known role in promoting metastasis, thereby promoting tumorigenesis ⁹². Based on this collective evidence, miRNA reprogramming of both the cancer cells and MSCs plays a role in the regulation of tumorigenesis.

Direct communication between the tumor micro-environment and cancer cells through transfer of miRNAs

Exosomes (and other microvesicles) provide the means for both the micro-environment and cancer to alter epigenetics within the tumor through the direct transfer of miRNAs ⁹³. This direct transfer introduces miRNAs that subsequently target and functionally alter the cells they enter.

Several studies have highlighted the transfer of miRNAs by exosomes from cancer cells to endothelial cells to promote angiogenesis and metastasis. In a murine breast cancer model, miR-210 was found to be upregulated in cancer derived exosomes, promoting endothelial tubing and branching ⁹⁴. This transfer of miR-210 through exosomes promoted tumorigenesis through increased endothelial cells in the TME ⁹⁴. Furthermore, exosomes from a human derived breast cancer cell line upregulated expression of miR-105 in endothelial cells, promoting metastasis to the lungs and brain ⁹⁵. The exosomes reduced endothelial barriers through miR-105 targeting of ZO-1 ⁹⁵. miR-105 was also found to be elevated in exosomes isolated from the serum of breast cancer patients ⁹⁵. Similar observations were made in multiple other types of cancers. Through coculturing 5 different tumor cell lines including non-small cell lung cancer, glioblastoma, pancreatic cancer, colorectal cancer and melanoma with endothelial cells and performing a miRNA array on the endothelial cells miR-9, miR-96, miR-182, and miR-183 were found to be consistently upregulated ⁹⁶. Specifically, cancer mediated secretion of miR-9 in exosomes promoted migration and in vivo endothelial density, which, in turn, promoted tumor growth ⁹⁶. The upregulation of miR-9 in endothelial cells through exosome delivery promoted angiogenesis through targeting SOCS5, resulting in increased STAT1 and STAT3 activation ⁹⁶. In glioblastoma, several well-known miRNAs that are highly expressed (let-7a, miR-15b, miR-16, miR19b, miR-21, miR-26a, miR-27a, miR-92, miR-93, miR-320, and miR-20) were found in the microvesicles isolated from patients ⁹⁷. Furthermore, these microvesicles promoted endothelial tube formation in an RNA dependent manner suggesting that the miRNAs in these microvesicles advance angiogenesis ⁹⁷. Additionally, it appears that cancers may downregulate miRNAs to limit their incorporation into exosomes that would constrain tumorigenesis. An example of tumor inhibition from miRNA transfer through exosomes is the transfer of miR-1 in microvesicles resulting from the reconstitution of miR-1 in glioblastoma, which acted to reduce tumor growth ⁹⁸. The upregulation of miR-1 in endothelial cells reduced endothelial cell recruitment and branching through miR-1 targeting of ANXA2 (annexin A2), which is highly expressed in some cancers and promotes tumorigenesis ^{98, 99}. Interestingly, miRNA composition varied between renal cancer stem cells and renal cancer cells. By utilizing a miRNA array of microvesicles from renal cancer patients and sorting cancer stem cells with CD105 expression it was shown that 24 miRNAs were upregulated and 32 miRNAs downregulated in renal cancer stem cells vs. renal non-cancer stem cells ¹⁰⁰. Furthermore, these microvesicles promoted endothelial adhesion and invasion in an RNA dependent manner, illustrating the potential for differences in miRNA composition of exosomes within the same tumor from different types of cancer cells ¹⁰⁰. The transfer of miRNAs to endothelial cells is not limited to solid cancers. Exosomes from leukemia upregulated expression of the miR-17-92 family in endothelial cells during co-culture and promoted endothelial branch formation and migration ¹⁰¹. Collectively, these studies demonstrate that cancer cells directly alter the miRNA composition of endothelial cells through exosomal transfer of cancer-derived miRNAs to modify the TME. These cancer-mediated changes in miRNA expression in endothelial cells promote angiogenesis and tumor invasion.

miRNA transfer via exosomes from cancer is not restricted to endothelial cells. Fibroblasts, bone marrow derived stromal cells, and macrophages uptake exosomes from cancer, which leads to the upregulation of cancer-derived miRNAs that alter the function of these cells. In a prostate cancer model, “oncosomes” isolated from prostate epithelial cells transformed with Kras infection showed increased levels of miR-1227. The oncosomes were taken up by CAFs, which induced their mobility ¹⁰². A miRNA array of exosomes isolated from a melanoma cell line identified 154 miRNAs within the exosomes ¹⁰³. Bone marrow stromal cells in co-culture took up these exosomes, which increased migration ¹⁰³. Cancer produced exosomes also regulate the immune system to promote tumorigenesis through TLR signaling. Exosomes from lung cancer cell lines were shown to bind to human TLR8 and to murine Tlr7 which is critical for tumorigenesis in a murine lung cancer model ¹⁰⁴. Specifically, miR-21 and miR-29a were secreted in exosomes from lung cancer cell lines and bound to TLR8 ¹⁰⁴. Additionally, miR-29 was shown to co-localize with exosome markers in human lung cancer samples ¹⁰⁴. Therefore, the evidence indicates that cancer cells directly reprogram stromal cells to support cancer progression through miRNA transfer by means of exosomes. While more is known about the secretion of exosomes from cancer cells and their effects on tumorigenesis, stromal cells, including macrophages and MSCs, secrete exosomes that modulate miRNA expression in cancer cells, thereby regulating tumorigenicity. IL-4 activated macrophages, promoting an M2 phenotype, were shown to secrete exosomes containing miR-223. ¹⁰⁵. The macrophage derived exosomes upregulated miR-223 during co-culture with a breast cancer cell line, which promoted breast cancer invasiveness ¹⁰⁵. Exosomes secreted by MSCs isolated from the bone marrow of patients with multiple myeloma promoted proliferation and metastasis. However, MSCs from healthy patients inhibited tumorigenesis in multiple myeloma. When the miRNA composition of the exosomes from MSCs taken from multiple myeloma patients was compared with that from healthy patients, 16 miRNAs were found to be downregulated and 2 upregulated ¹⁰⁶. Interestingly, miR-15, a known tumor suppressing miRNA, was downregulated in exosomes from MSCs isolated from multiple myeloma patients ¹⁰⁶. This downregulation of miR-15, along with other miRNA changes within exosomes, resulted in protumorigenic effects of exosomes ¹⁰⁶. In a model of gastric cancer, miR-214, miR-221, and miR-222 were shown to be highly upregulated and MSCs were found to transfer miR-221 via exosomes ¹⁰⁷. However, MSCs also reduce tumorigenesis through exosomal secretion. In a murine breast cancer model, exosomes from MSCs transferred miR-16 to breast cancer cells reducing angiogenic capacity ¹⁰⁸. miR-16 reduced angiogenesis through targeting VEGF, interfering with the ability of breast cancer cells to promote endothelial branching, migrate, and increase microvessel density ¹⁰⁸.

Transfer of miRNAs between cells is not limited to microvesicle packaging. It has been demonstrated that miRNAs are transferred through direct cell contact via gap junctions, functionally altering protein expression through miRNA targeting of the mRNA. A recent study that may be relevant to our understanding of the immune micro-environment showed that miRNAs from macrophages were transferred to HCC cells reducing proliferation ¹⁰⁹. Specifically, miR-142 and miR-223, which are highly expressed in macrophages, are transferred to HCC cells through direct cell contact mediated by gap junctions ¹⁰⁹.

Conclusion

This review highlights the emerging relevance of miRNAs in the communication between cancer cells and the TME. The addition of another level of complexity, represented by the interactions between cancer and stromal cells involving the regulation of miRNAs, presents yet another obstacle to a comprehensive understanding of tumor growth and progression, while also providing new and promising therapeutic targets. While there are many cancer specific deregulations of miRNA in both the cancer cells and cells of the TME there are a few miRNAs that have been reported to be deregulated in TME components in more than one cancer (Figure 5). These could play a more fundamental role in the bidirectional communication between cancer cells and the TME.

Figure 5.

miRNA changes in the components of the TME that occur in more than one cancer. References: [1], ⁴⁰; [2], ⁴¹; [3], ⁴²; [4], ⁴³; [5], ⁴⁴; [6], ⁵²; [7], ⁴⁸; [8], ⁵⁰; [9], ²¹; [10], ⁶². CAF, cancer associated fibroblast; CAEs, cancer associated endothelial cells.

The importance of miRNAs and their ability to reprogram cells to regulate function within the TME will be further elucidated, as our understanding of miRNA biology progresses and the complicated network of the TME is revealed. The studies in this review confirm that there is an active communication between cancer cells and the stroma mediated through miRNA changes. However, the extent of this communication has yet to be fully explored. Currently, the majority of the miRNA changes detected in the TME are dictated by the cancer cells. However, there is plenty of evidence that endothelial cells, fibroblasts, and infiltrating immune cells influence tumorigenesis. It is likely that we will find that miRNAs are significant factors in the influence of stromal cells on cancer progression

Clinically altering gene expression and targeting RNAs has been met with limited success, However it is an active area of research. The primary complication with RNA targeting therapies, like most cancer therapies, is specificity for the TME and cancer cells. Understanding how cancer cells communicate with the stroma within the TME, especially through exosomes, may uncover an avenue for specific targeting. The specificity, packaging, and proximity of miRNA containing exosomes within the TME are still unknown. However, these features of exosomes are likely be actively explored, since understanding them could lead to distinct clinical benefits. If we can determine if certain miRNA containing exosome are destined to be taken up by specific cells, we could provide a mechanism and pathway for specific cell targeting, allowing for delivery of chemotherapeutics and gene therapies.

That miRNAs and proteins are not randomly packaged within exosomes is evidenced by the differences in proportions of miRNAs contained with exosomes as compared to the overall cell content ^{97, 110-115}. The miRNA content of exosomes isolated from the peripheral blood from OvCa patients is significantly altered from that of healthy patients, suggesting that the miRNA profile of exosomes might be used for diagnostics ¹¹⁶.

The question remains whether the exosomes that are found in the blood accurately correspond to the exosomes released in the TME. While cancer cells are thought to produce the majority of miRNA containing exosomes, as illustrated in this review, endothelial cells and immune infiltrating cells secrete exosomes in the TME as well. It is possible that once the communication between cancer cells and the stroma is decoded, these miRNA communication pathways may be exploited in strategies for early cancer detection, monitoring therapeutic responses, or, potentially, for targeted cancer therapy.

Abbreviations

BRCA-1

breast cancer 1, early onset

VE-cadherin

vascular endothelial cadherin

MAZ

Myc-associated zinc finger protein

HGF

hepatocyte growth factor

IGFBP2

Insulin-like growth factor-binding protein 2

PITPNC1

phosphatidylinositol transfer protein, cytoplasmic 1

MERTK

c-mer proto-oncogene tyrosine kinase

VHL

von Hippel-Lindau tumor suppressor, E3 ubiquitin protein ligase

LATS2

large tumor suppressor kinase 2

APOE

apolipoprotein E

DNAJA4

DnaJ (Hsp40) homolog, subfamily A, member 4

MMP2

matrix metallopeptidase 2

VEGF

vascular endothelial growth factor

HIF1A

hypoxia inducible factor 1, alpha subunit

FLT1

fms-related tyrosine kinase 1

IL

interleukin

VEGFR

vascular endothelial growth factor receptor

TGFB

transforming growth factor beta

ETS2

v-ets avian erythroblastosis virus E26 oncogene homolog 2

MMP9

matrix metallopeptidase 9

EMILIN2

elastin microfibril interfacer 2

CCL5

chemokine (C-C motif) ligand 5

SATB2

Special AT-Rich Sequence-Binding Protein 2

WNT10B

wingless-type MMTV integration site family, member 10B

PTEN

phosphatase and tensin homolog

SOCS1

suppressor of cytokine signaling 1

SDF1

stromal cell-derived factor 1

STAT3

signal transducer and activator of transcription 3

CEBPB

CCAAT/enhancer binding protein (C/EBP), beta

IRF4

interferon regulatory factor 4

TME

Tumor Micro-environment

CAF

cancer associated fibroblasts

ECM

extracellular matrix

CXCR4

CXC chemokine receptor 4

TNF- α

tumor necrosis factor alpha

NF- κ B

nuclear factor-kappa B

iNOS

Inducible nitric oxide synthase

MHC

major histocompatibility complex

iPS

induced pluripotent stem cells

HCC

hepatocellular carcinoma

PDGFRA

platelet-derived growth factor receptor, alpha polypeptide

EGF

epidermal growth factor

FGF

fibroblast growth factor

GAS6

growth arrest-specific 6

GATA3

GATA binding protein 3

ITGA6

integrin, alpha 6

ANGPTL4

angiopoietin-like 4

LOX

lysyl oxidase

TCGA

the cancer genome atlas

EMT

epithelial-mesenchymal transition

MDSC

myeloid derived suppressor cell

MAPK

mitogen-activated protein kinase

AKT

v-akt murine thymoma viral oncogene homolog

ERK

extracellular-signal-regulated kinases

OvCa

ovarian cancer

DNMT1

DNA methyl transferase 1

uPA

urokinase-type plasminogen activator

TAM

tumor associated macrophage

IFN γ

interferon gamma

Th1

T helper 1

Fra-1

Fos-related antigen

AP1

adaptor-related protein complex 1

CTBP2

C-terminal binding protein 2

SOX2

SRY (sex determining region Y)-box 2

MSC

mesenchymal stem cell

ZEB1

zinc finger E-box binding homeobox 1

ZEB2

zinc finger E-box binding homeobox 2

ZO-1

zonula occludens

ANXA2

annexin A2

TLR

toll like receptor

CCL-22

chemokine (C-C motif) ligand 22

FGF11

fibroblast growth factor 11

ICAM-1

intercellular adhesion molecule 1

BLIMP-1

B-lymphocyte-induced maturation protein 1