Remodeling the Microenvironment before Occurrence and Metastasis of Cancer.

Tumorigenesis and progression of cancer are complex processes which transformed cells and stromal cells interact and co-evolve. Intrinsic and extrinsic factors cause the mutations of cells. The survival of transformed cells critically depends on the circumstances which they reside. The malignant transformed cancer cells reprogram the microenvironment locally and systemically. The formation of premetastatic niche in the secondary organs facilitates cancer cells survival in the distant organs. This review outlines the current understanding of the key roles of premalignant niche and premetastatic niche in cancer progression. We proposed that a niche facilitates survival of transformed cells is characteristics of senescence, stromal fibrosis and obese microenvironment. We also proposed the formation of premetastatic niche in secondary organs is critically influenced by primary cancer cells. Therefore, it suggested that strategies to target the niche can be promising approach to eradicate cancer cells.

Introduction

It is widely accepted that tumorigenesis is a multistage process during which molecular alterations in the genome of somatic cells accumulate. Gene mutations force normal cells to grow abnormally. Although most DNA replicates with fairly high fidelity, mistakes do happen1. However, the link between mutation and cancer incidence appears to be more complex2. People also observed that only a fraction of cells within tumors were capable of clonogenic growth. The heterogeneity among cancer cells can arise in multiple ways. Two theories have been proposed to explain this heterogeneity: extrinsic factors and intrinsic factors3. Evolutionary theories are always applied to understand how cancers develop and how heterogeneity exists. In this way, carcinogenesis is viewed as a Darwinian process of successive rounds of selection leading to the accumulation of mutations4, 5. Cells face diverse selective pressures as they react to changes in their environment6. The Darwin theory of evolution has been determined by the match between the current environmental demand and the phenotypic manifestation of mutations5. The competitive advantage of mutations during tumor initiation is dependent on the context in which they arise. Below, we will focus on cooperative relationship between transformed cells and microenvironment on the initiation and development of cancer (Figure 1).

Figure 1

Schematic diagram of formation of the premalignant niche and premetastatic niche. Mutations result either from DNA replication errors or from the damaging events. Accumulation of unrepaired mutations transforms normal cells. The survival of transformed cells critically depends on the circumstances which they reside. The niche at high risk of malignant transformation is associated with aging, fibrosis and obesity. Bone marrow-derived cells (BMDC) including haematopoietic progenitors, mesenchymal stem cells, endothelial progenitor cells comprise the main component in the premetastatic niche. Tumor-derived secreted factors (TDSFs) are crucial in creating a supportive microenvironment at the metastatic site. Chemokines or cytokines derived from the primary cancer cells reprogramming the distant organs and contribute to the establishment of premetastatic niche. Exosomes participate in cell-to-cell communication by the molecules enriched in their membrane, remodeling the microenvironment of target organs and help the formation of premetastatic niche.

Premalignant niche

Mutations result either from DNA replication errors or from the damaging events. Accumulation of unrepaired mutations transforms normal cells. The survival of transformed cells critically depends on the circumstances which they reside. The niche at high risk of malignant transformation is associated with aging, fibrosis and obesity.

Senescence-messaging secretome (SMS)

Aging is the biggest risk factor for cancer. By 2030, 70% of the tumor will occur in the population of 65 years and older7. In humans, cancer incidence rises with approximately exponential kinetics after 50 years of old8, partly due to accumulation of oncogenic mutations over time. Cellular senescence, which is associated with aging, is a state of irreversible growth arrest9. It was previously assumed that senescence was functionally similar to apoptotic cell death10. However, senescent cells show marked and distinct changes in their pattern of gene expression 8, 11. It is reported that tissue microenvironment is the main cause of the occurrence of age-related tumors 12-14. The senescence-associated secretory phenotype (SASP; also known as the senescence-messaging secretome (SMS)) provides senescent cells with diverse functionality10. The nature of the SMS and its targets, and the overall downstream outcomes, vary considerably depending on the cellular context10. On the one hand, SMS can aid tissue repair, but on the other hand, the SMS can do great damage to normal tissue structures and function, promote malignant phenotypes in nearby cells8. The SMS contains several families of factors that can be divided into the following 3 major categories: soluble signaling factors (interleukins, chemokines, and growth factors), secreted proteases, and secreted insoluble components9. These SMS accumulate with age that may change the tissue microenvironment and promote the occurrence of chronic inflammatory diseases or tumors. The most prominent soluble factors are Interleukin-1 (IL-1) and Interleukin-6 (IL-6). IL-1 and IL-6 have been proposed as major upstream regulators of the senescence-associated cytokine network10. Circulating IL-1 and IL-6 promote a variety of chronic degenerative diseases, as well as cancer15-23. There is little measurable IL-6 in the circulation in the absence of inflammation. With advancing age, however, serum IL-6 becomes detectable even without evidence of inflammation. It is proposed that this reflects an age-associated loss in the normal regulation of gene expression for this molecule. There is also speculation that IL-6 may contribute to the pathogenesis of several diseases that are common in late-life including lymphoma, osteoporosis, and Alzheimer's disease24, 25. Senescent fibroblast cells also express IL-8, GROα, GROβ, MCP-2, MCP-4, MCP-3, MCP-1, MIP-3α, MIP-1α, CCL-1, IGFBPs, GCSF, GM-CSF9. Aside from soluble signaling cytokines and growth factors, senescent cells also secrete increased levels of matrix metalloproteinases (MMPs) and other molecules such as reactive oxygen species(ROS), nitric oxide and transported ions.

Fibrotic niche

Pathologic fibrosis is the feature of abnormal extracellular matrix (ECM) deposition caused by prolonged injury and deregulated processes of wound healing. Extracellular matrix proteins including collagen, elastin, fibronectin and laminin are abundant in ECM. Fibrotic diseases encompass a wide spectrum of entities including idiopathic pulmonary fibrosis (IPF),silicosis, asbestosis, ischemic heart disease, cirrhosis, splenic fibrous hyperplasia et al. It has long been observed that fibrosis is related to carcinogenesis. In some aspects, a tumor can be viewed as a fibrotic organ that contains cancer cells. A tumor cannot develop without the parallel expansion of a tumor stroma26. Fibroblasts are involved in tissue remodeling and repair. Physiologic fibroblasts maintain stromal homeostasis. Pathologic fibrosis always began with reaction to inflammation, characterized by pathologic fibroblasts and a stiff ECM27. Transforming growth factor-β (TGF-β) is the most predominant profibrotic growth factor, leading to an increase of collagen and fibronectin production by fibroblasts and the transition of fibroblasts into myofibroblasts. TGF-β also activates other stromal cells such as hepatic stellate cells (hStCs) to produce fibronectin (FN).

The increased rigidity of ECM is an important determinant for cell behavior28, 29. Substrate rigidity influences cell morphology, cell migration and cell growth28. Upon sensing force, cells react by active change in the actin cytoskeleton. The rigidity of ECM regulates localization and activity of YAP/TAZ30. The process of Epithelial-Mesenchymal-Transition (EMT) is known to result in a phenotype change in cells which are in a more invasive state31. Mechanical stress induces EMT by both physical forces and biochemical signals32, 33. Mechanical stress induces Twist expression in a manner dependent on β-catenin34. Recent studies highlight a link between EMT and cancer stem cell formation35.

In denser, stiffer matrices (∼44 kPa), murine mammary epithelial cells displayed more invasive phenotypes, compared to the softer matrices of lower density (∼25 kPa)36. Pressure to osteocytes, the main mechanotransducing cells in bone, induces prostate cancer growth and invasion29.The mechanical microenvironment may cause malignant transformation, possibly through stimulating intracellular signaling pathways that promote cancer cell survival or invasion28, 37. Matrix density-induced stiffness regulates epithelial cell phenotype, promotes cellular adhesion through a FAK-ERK signaling36. Transmembrane cell adhesion proteins, mostly integrins link the extracellular matrix to the cell's cytoskeleton. Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages38. Besides the roles in cell adhesion, integins have an activating role in intracellular signaling events as signaling receptors. The different heterodimer with alpha and beta subunits cause the functional and molecular diversity of integrins. Upon integrin aggregation, a number of signal transduction pathways, such as the Raf-ERK/MAPK, PI3K-Akt, nuclear factor-kappa B (NF-κB), and c-Jun are activated39.

In addition, substrate stiffness modulates genes expression, such as focal adhesion proteins (Itga6 and Parvb), cytoskeletal proteins (Dnah11 and Actb), and nuclear envelope protein Lbr and Nrm 40.The mechanisms how mechanical stresses affect the expression of genes that influence cell adhesion, migration can be explained by nucleus shape or LINC-mediated linking between cytoskeleton to the nucleoskeleton40, 41. Dynamic force-induced structural changes in Cajal bodies, a prominent nuclear body, may affect nuclear functions involved in gene expression42. Mechanical stresses can be transmitted from the cytoskeleton to the nucleus by LINC (linker of nucleoskeleton and cytoskeleton) complex that is composed by SUN1/2 proteins and regulates gene expression 40, 43.

Obesity niche

More and more evidence demonstrated obesity is linked to the increased risk of cancer incidence and mortality44. Obesity is excess fat in the body45. Obesity is closely related to type 2 diabetes, hyperlipidemia, hypertension and cardiovascular and cerebrovascular diseases45-47. Obesity is quickly overtaking tobacco as the leading preventable cause of cancer. Obesity contributes to the occurrence and development of cancer systemically or locally through affecting energy imbalance including insulin resistance, altered hormone signaling, and high circulating levels of proinflammatory mediators44. Hyperadiposity as a result of excess caloric intake or reduced caloric expenditure cause production of steroid hormones and adipokines. Adipose tissue has been considered to be the largest endocrine organ in the body, producing adipokines, cytokines and chemokines involved in metabolism and immune regulation44-53. A crosstalk between estrogen, insulin, insulin-like growth factor-1 (IGF-1) and adipokine signaling pathways plays an important role in the development of cancer51. Adipokines, including leptin and adiponectin, are hormones produced by adipocytes. There are conflicting data regarding the roles of adiponectin in the development of cancer. Circulating plasma concentrations of Adiponectin are inversely related to increased risks of malignancy. Decreased level of adiponectin are present in patients with breast cancer, prostate cancer, gastric cancer, et al54. Adiponectin inhibits cancer cell proliferation and promotes cancer cell apoptosis through inhibiting STAT3, PI3K/AKT, Wnt signaling54. However, increased adiponectin have been associated with increased risk of lung cancer and hepatic cancer55, 56. higher levels of adiponectin or higher adiponectin/leptin ratios in pancreatic cancer patients with positive or strongly positive expression of adiponectin receptor 1 and receptor 257, 58. Adiponectin is also reported to have proliferative effect on cancer cells through enhancing ceramide catabolism and anti-apoptotic metabolite S1P59. Leptin and its receptor OB-R have been implicated in a number of malignancies through activating Janus kinase/Signal transducer and activator of transcription (JAK/STAT), PI3K/Akt and extracellular regulated protein kinases(ERK). Leptin increases the expressions of the tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1alpha (HIF-α). It improves the ability of tumor cells to resist apoptosis, angiogenesis and hypoxia tolerance, which is beneficial to the progression and metastasis of tumor60-62. Besides, proinflammatory mediators such as C- reactive protein (CRP), TNF-α, IL-6, IL-8 in the circulation which is produced by adipocytes promote neoplasia and tumor progression locally and systemically63. Obesity-induced interstitial fibrosis promotes breast tumorigenesis by altering mammary ECM mechanics with important potential implications for anticancer therapies64. Insulin resistance is associated with worse prognosis in several cancers and insulin can stimulate the synthesis of IGF-1, which is linked to tumor progression. Insulin and IGF-1 activate the PI3K/Akt/mTOR and Ras/Raf/ MAPK pathways65-70. Obesity enhances local myofibroblast content in mammary adipose tissue and that these stromal changes increase malignant potential by enhancing interstitial ECM stiffness64.

Suitable microenvironment for cancer progression

Premetastatic niche

Metastasis is the most life threatening event in cancer patients71. Metastasis can occur when cells break away from a primary tumor and travel through blood stream or through lymph vessels to other areas of the body, which is responsible for approximately 90% of cancer deaths72. The earlier theories regarded metastasis as a process of orderly anatomic spread73. In contrast, Fisher hypothesized that whether distant relapse occurs is predetermined from the onset of tumorigenesis74. It has been also noticed clinically that cancer metastasis does not scale with primary tumor size. Circulating cancer cells can be detected in varied cancers. However, metastasis is largely an inefficient process in which most circulating cancer cells fail to mature in a clinically meaningful fashion75. Researchers have identified dormant cancer cells in metastasis-free organs76. Disseminated tumor cells were detected in 30% of patients diagnosed with early stage (I-III) breast cancer, however, patients with disseminated tumor cells did not uniformly develop metastatic disease77. In 1889, Dr. Stephen Paget stated that metastasis did not occur randomly. His study which is published in The Lancet, demonstrated that metastasis only develop when the seed and soil are compatible. In this respect, tumors may have greater or lesser ability to colonize lymph nodes and distant organs, as driven by their match to the distant microenvironment78. Such permissive environment is always formed before arrival of cancer cells, which is called “premetastatic niche”79.

Premetastatic niche is also composed of a heterogeneous mixture of stromal cells, vasculature, other supportive cells and extracellular matrix75. Bone marrow-derived cells (BMDC) including haematopoietic progenitors, mesenchymal stem cells, endothelial progenitor cells comprise the main component in the premetastatic niche. Bone marrow-derived cells (that is, macrophages and granulocytes) bind to FN-enriched hepatic sites, ultimately leading to liver pre-metastatic niche formation80. Metastatic cancer cells which overexpress Jagged activate both haematopoietic osteoclasts and mesenchymal osteoblasts by binding to Notch and promote both tumor cell growth and invasion in the bone. Tumor-derived secreted factors (TDSFs) are crucial in creating a supportive microenvironment at the metastatic site. Chemokines or cytokines derived from the primary cancer cells reprogramming the distant organs and contribute to the establishment of premetastatic niche81. Besides chronic inflammation, acute inflammation in the lung can foster metastatic seeding82, 83. Bacteria- and LPS-induced acute inflammation significantly enhanced lung metastasis. Acute lung infection dramatically increased cancer cell homing to the lung. A large number of the recent exosome literature highlights the roles of cancer cell-derived or stroma cell-derived exosomes on the reprogramming of microenvironment80, 84, 85. Exosomes are small, 30 to 100-nm membrane vesicles formed by the inward budding of late endosomes86-88. Exosomes contain cytokines, transcription factor, growth factor, and other bioactive molecules such as miRNA, LncRNA et al 89-91. They participate in cell-to-cell communication by the molecules enriched in their membrane, remodeling the microenvironment of target organs and help the formation of premetastatic niche 80, 92-102(Table 1). Exosomes are widely distributed in various human body fluids, such as blood plasma/ serum, saliva, breast milk, cerebrospinal fluid and urine. Exosomes that are enriched with cancer-specific miRNAs, LncRNAs can be used as biomarkers for cancer progression 103-107.

Table 1

Exosomes and the formation of premetastatic niche

Diseases	Exosomal moleculres	Type	Source cell	Target cell	Mechanisms	Reference
PDAC	MIF	protein	PDAC cells	macrophages	Fibrotic niche	80
BCa	miR-122	miRNA	BCa cells	fibroblasts	Glucose metabolism niche	92
PC	miR-301a-3p	miRNA	PC cells	macrophages	Inflammatory niche	93
HCC	miR-103	miRNA	Hepatoma cells	Endothelia	Angiogenesis niche	94
NPC	miR-23a	miRNA	NPC cells	endothelia	Angiogenesis niche	95
HCC	miR-1247-3p	miRNA	HCC cells	fibroblasts	Fibrotic niche	96
Gastric cancer	EGFR	protein	gastric cancer cells	stromal cells	Fibrotic niche	97
BCa	miR-23b	miRNA	MSCs	BCa cells	dormancy niche	98
HCC	miR-210	miRNA	HCC cells	endothelial	Angiogenesis niche	99
Colon cancer	CEACAMs	protein	Colon cancer cells endothelial	T-cells	Immunosuppression niche	100

Notes: PDAC: Pancreatic ductal adenocarcinomas; MIF: macrophage migration inhibitory factor; BCa: breast cancer; PC: pancreatic cancer; HCC: hepatocellular carcinoma.NPC:nasopharyngeal carcinoma;EGFR:epidermal growth factor receptor;MSC:mesenchymal stem cell; CEACAMs: carcinoembryonic antigen related cell adhesion molecules;

There are 6 characteristics and traits that define pre-metastatic niche including immunosuppression, inflammation, high angiogenesis and vascular permeability, active lymphangiogenesis, specific organotropism and high reprogramming efficiency81.

Angiogenesis niche

Formation of vascular network is important to the proliferation and dissemination of cancer cells. Stable microvessels form a "dormant niche". Factors that sustain the homeostasis such as endothelial-derived thrombospondin-1 induce sustained cancer cells quiescence. When blood vessels begin to sprout, the new tips produce molecules that transform dormant cancer cells into metastatic tumors108, in which process the thrombospondin-1 proteins give way to tumor necrosis factor (TNF) and periostin proteins in the neovasculature. Many proinflammatory chemokines generated by cancer cells support the development of vessels. TNF-α acts indirectly by inducing the production and release of VEGF and bFGF109, 110. CXCR4 promotes the migration of endothelial cells toward stromal cells derived factor SDF-1 to branch and develop new vessels. SDF-1-CXCR4 interaction increases VEGF production by endothelial cells, and VEGF and bFGF in turn enhances SDF-1109, 110. VEGFR1+VLA-4+ haematopoietic progenitors move from bone marrow and home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells. These haematopoietic progenitors dictate organ-specific tumour spread through angiogenesis and chemotaxis.

Immunosuppression niche

Cancer cells somehow are like “foreign” material. Immune system recognizes and eliminates the cancer cells during the early phase of cellular transformation. The process of tumor immuno-editing includes the following three key phases: elimination, equilibrium, and escape111. During the escape phase, cancer cells resist the selective pressure from the immune system by acquiring mutations or undergoing other changes that allow for tumor progression in the face of an ongoing immune response111-114. The escaped cancer cells shape the immune system to be immunosuppressive to allow themselves to grow. Myeloid-derived suppressor cells (MDSC) and regulatory Treg cells are major components of the immune suppressive cells. These suppressor cells alter the microenvironment through the secretion of inflammatory and immunosuppressive cytokines to promote metastasis. There is an accumulation of metabolic enzymes that suppress T cell proliferation and activation, including IDO and arginase, and high expression of tolerance-inducing ligands like FasL, PD-1, CTLA-4, and B7115-117. Tumor-derived vesicles known as exosomes have also been implicated promoting differentiation of iTreg cells and myeloid derived suppressor cells (MDSCs)118. Suppressive immune cell populations such as Gr1+CD11b+ myeloid cells at secondary organ sites increase regional inflammatory cytokines such as S100A8 and S100A9 that promotes metastatic seeding119, 120. Primary tumor induction of S100A8 and S100A9 expression has also been shown to recruit Mac1+ myeloid cells via TLR4 to premetastatic sites121, 122.

Directional movement of cancer cells to the premetastatic niche

Homing to and seeding in the secondary sites are the most important steps in the process of metastasis. Homing is a rapid process in which cancer cells actively cross the blood/endothelium barrier by adhesion interaction. Cell adhesion molecules (CAMs) are glycoproteins synthesized by cells, which are involved in interaction between cells and cells or cells and matrix, and involve in cell signal transduction and cell migration. CAMs can be divided into four main groups: selectins, integrins, Ig superfamily and cadherins. Integrins are heterodimeric transmembrane adhesion receptors that bind to ECM ligands outside a cell to the actin cytoskeleton inside the cell123. There are several types of integrins on the cell surface. The binding specificity allows cells expressing certain integrin heterodimers pass through an ECM containing specific components124. Cells sense and respond to their environment through spatio-temporal patterns of integrin versus ligand expression124. Ligands of integrins are fibronectin, vitronectin, collagen, and laminin. Integrins play physiological or pathological roles depending on the components of ECM. Integrin αvβ5, α5β1, α6β4, α9β1 on the cell surface help to maintain normal homeostasis through binding to fibronectin TSP-1. Stromal compartment initially inhibit cancer progression by maintaining architecture125. Alterations in integrin including aberrant expression and activation of downstream effectors are involved in carcinogenesis39. Most circulating cancer cells die in circulation as a result of shear stress and/or anoikis126. Activated αvβ3 can keep circulating cancer cells from shear stress by binding to leukocytes and platelets127. When circulating cancer cells once arrive at distant organs, integrin-ligand interactions help cancer cells colonize to the metastatic environment39. Cancer cell invasion are heavily dependent on integrin-mediated adhesion to the ECM128. Intergrin α4β1 permits cancer cell engagement of fibrinogen, ICAM and VCAM are expressed by the vascular and stromal cells of bone marrow129. Integrin α5β3 on cancer cells not only helps the cancer cells home to bone marrow through adhering to the vitronectin, osteopontin, bone sialoprotein, fibronectin and thrombospondin in bone marrow but also serves as physical anchors permitting metastatic cells to establish footholds in the bone marrow. α6β4, α6β1 and αvβ5 have been showed to be expressed in exosomes and mediated lung cancer cell metastasis130. Exosomal integrins can activate the phosphorylation of Src and the expression of the pro-inflammatory S100130. Cancer cell invasion can occur as individual cell migration or collective cell migration, both of which are dependent on downregulation of E-cadherin induced loosening of cell junction128. The crosstalk between integrins and E-cadherin mediate epithelial cell-cell adhesion and cell-matrix adhesion signaling. The balance between E-cadherin-mediated adhesion junctions and integrin-mediated cell-matrix contacts determine the metastasis process. α-catenin has a pivotal role in the crosstalk between E-cadherin adhesions and integrin-mediated cell-ECM interactions131.

Tumor cell homing to secondary organs is regulated by cytokines, chemokines, and their receptors 78. The chemokines, such as CXCL12, have been demonstrated a driving role in the directional movement of cancer cells which overexpressed CXCR4132. Lungs, bone, liver, brain, and regional lymph nodes that express high levels of stromal cell-derived factor-1 (SDF-1α/CXCL12) are the most common sites for residence of breast cancer cells expressing CXCR4133, 134. CXCL12 is also a ligand that promotes chemotaxis of endothelial cells and hematopoietic progenitors to bone marrow from circulation135, 136. Factors including VEGF-A, TGF-β, and TNF-α attract tumor cells by upregulating the expression of S100A8 and S100A9137.

Conclusion

Tumorigenesis and progression of cancer are complex processes which transformed cells and stromal cells interact and co-evolve. Intrinsic and extrinsic factors cause the mutations of cells. A niche in high-risk localized cancer cells is characteristics of senescence, stromal fibrosis and obese microenvironment which contribute on the survival of mutated cells. Cancer cells reprogram the microenvironment locally and systemically. The formation of premetastatic niche in the secondary organs facilitate the cancer cells survival in the distant organs. Strategies to target the niche can be promising approach to eradicate cancer cells.