Adipocyte regulation of cancer stem cells

1 INTRODUCTION

Interactions between tumor cells and the associated stroma, called tumor microenvironment (TME), represent a powerful relationship that influences disease initiation, progression, and patient prognosis. Adipocytes are a component of TME which is composed of a variety of cells, including fibroblasts, infiltrating inflammatory cells, vascular endothelial cells, lymphatic cells, and neural cells,¹ and those that promote tumor proliferation, invasion, and immune suppression are called cancer-associated adipocytes (CAAs).² In the phenomena called adipocyte–mesenchymal transition (AMT), tumor cells induce the dedifferentiation of adipocytes into multiple cell types, including myofibroblast- and macrophage-like cells.³ These AMT cells become involved in various biological processes, such as immune response, inflammation, and extracellular matrix (ECM) remodeling and regulate tumor progression. AMT is at least partly induced by tumor-derived soluble factors, such as TNF-α, plasminogen activator inhibitor 1, Wnt3a, IL-6, IL-1β, and TGF-β, and exosomal microRNAs.

Adipose tissue is not simply the primary site for energy storage but functions as an active endocrine organ in which adipocytes secrete cytokines and hormones called adipokines. Adipokines play numerous roles in tumor formation and progression via modulating oncogenic signaling, metabolism, angiogenesis, and immune microenvironment.^4-6 Furthermore, lipids such as fatty acids (FAs) and FA derivatives, including phospholipids, sterols, and sphingolipids, secreted from adipocytes modulate processes ranging from the ECM remodeling to cancer cell signaling.⁷ Many cancers, such as colorectal, breast, prostate, lung, ovarian cancers, and hematologic malignancies, obtain FA from surrounding adipose tissue by stimulating lipolysis in adipocytes to promote energy metabolism (β-oxidation), membrane synthesis, and biosynthesis of lipid-derived cell-signaling molecules.⁸ Hypoxia in the adipose tissue of obese patients is caused by insufficient blood perfusion owing to a relatively low density of microvessels, which triggers angiogenesis, inhibits macrophage migration and preadipocyte differentiation, augments fibrosis, and suppresses recruitment of immune cells, all of which confer drug resistance abilities to cancer cells.⁹

Cancer stem cells (CSCs) are a highly tumorigenic subpopulation of cancer cells within a tumor and have a higher ability to promote tumor initiation^{10, 11} and metastatic progression, especially at the initial steps of metastases.¹² Therefore, CSCs are considered to be a critical target to ensure the complete elimination of tumor cells. Cell surface markers such as CD34, CD133, CD44, CD166, and CD24 and ALDH1 activity among others, have been operationally used to identify CSC populations.¹⁰ The purified CSC population is still heterogenous and some types of CSCs, like those of colorectal cancers (CRCs), exhibit high plasticity, allowing them to dynamically switch phenotype, such as stem/non-stem cell, epithelial–mesenchymal transition (EMT), and quiescent/proliferative condition, depending on stem cell niche and metabolic pathways of glycolysis and oxidative phosphorylation.¹³ Stem cells have a long-term self-renewal ability, a specific cellular action that involves proliferation accompanied by maintenance of both multipotency and tissue regenerative potential; and frequency and timing of actual stem cell self-renewal divisions are tightly regulated within the tissue to ensure the lifelong maintenance of the stem cell population. CSCs share several properties with normal stem cells from which they are derived, such as self-renewal ability and the ability to generate differentiated progenies. Epigenetic mechanisms, such as Polycomb gene complexes, histone modifications, microRNAs, and other noncoding RNAs, play important roles in the regulation of self-renewal and differentiation of both normal and malignant stem cells. We have revealed that targeting Polycomb group gene BMI1 by microRNA (miR)-200c is a shared molecular mechanism for stem cell regulation in both tissue stem cells and CSCs in breast and colon epithelium.^{14, 15}

The CSC niche maintains the properties of CSCs and facilitates their metastatic potential.¹⁶ We have proposed that mammary adipocytes function as a CSC niche in breast cancers and secrete adipsin (complement factor D, CFD) and its downstream effector HGF, resulting in the enhancement of CSC properties of breast cancer cells^{17, 18} (Figure 1). Understanding the essential roles of CSC niche in tumor initiation and progression will uncover novel strategies to treat cancers, especially those whose progression is linked to obesity.

2 CLASSIFICATION OF ADIPOSE TISSUES

Adipose tissue is classified into two main types: white adipose tissue (WAT) and brown adipose tissue (BAT), and WAT is further subdivided into subcutaneous adipose tissue (SAT) and visceral adipose tissues (VAT). A third and fourth types of adipocytes, beige and pink adipocytes, are generated by the transdifferentiation of SAT.

White adipose tissue, the most common type of adipose tissue in the body, is responsible for energy storage in the form of triglycerides, as well as adipokine secretion, hormone biosynthesis, insulation, mechanical protection of organs, and inflammation.¹⁹ WAT can be further subdivided into SAT and VAT. SAT is located just under the skin and throughout the body. It serves as an energy reserve and also provides insulation. VAT is found in the abdomen and around the internal organs. VAT is more metabolically active, contains more immune cells than SAT, and is associated with a higher risk of metabolic diseases like type-2 diabetes (T2D) and cardiovascular disease.

Brown adipose tissue is a specialized type of adipose tissue that generates heat to maintain body temperature. Thermoregulation by BAT utilizes uncoupling protein 1 (UCP1) that uncouples mitochondrial respiration from ATP synthesis. It is mainly found in infants and hibernating animals, but it also exists in small amounts in adults. Being rich in mitochondria, BAT is brownish in color and more metabolically active than WAT. BAT secretes a collection of active peptides called batokine, including FGF2, BMP8b, NRG4, IL-6, and IGF-1.²⁰

Beige adipocytes are interspersed within the WAT and can be induced by “browning” of WAT. Activated beige adipocytes are characterized by high levels of FA β-oxidation, mitochondrial content, and thermogenesis.²¹

Pink adipocytes are found in lactating mammary adipose tissue and are named after their distinctive pink appearance when stained with hematoxylin and eosin. During lactation, the mammary gland undergoes extensive remodeling, including an increase in adipose tissue to support milk production. Pink adipocytes are thought to play a role in providing energy substrates to support milk production, and emerging evidence suggests that they derive from the transdifferentiation of subcutaneous white adipocytes.²²

White adipose tissue and its association with various diseases, including cardiovascular disease, T2D, metabolic syndrome, and cancers, have been extensively studied. In contrast, the roles of BAT, beige and pink adipocytes in cancers are not fully elucidated. Activation of thermogenic metabolism in BAT facilitates tumor suppression by enhancing blood glucose uptake in adipocytes and impeding the glycolysis-based metabolism in cancer cells, which are alleviated by the removal of BAT or genetic deletion of UCP1.²³

3 ADIPOCYTES AND CSCs

The relationship between adipocytes and CSCs is complex, and the precise effects of adipocytes on CSCs can vary depending on the type of cancer and the particular context.

4 MOLECULAR MECHANISMS OF CSC REGULATION BY ADIPOCYTES

Adipocytes regulate the stem cell properties in the tissues, such as mammary tissue and bone marrow. However, the molecular mechanism for the stem cell and CSC niche function of adipocytes is not clearly demonstrated (Figure 2).

4.1 Adipokine

Adipokines released from the adipocytes contain more than 600 bioactive molecules that act in paracrine and endocrine manners and function as mediators of adipocyte–cancer cell interactions. Some of these adipokines are exclusively released from adipocytes, whereas the others are secreted from other tissues as well. Adipokine secretion is induced by various stimuli, including metabolic status, adipokines, inflammation, mechanical stress, and exosomes, which are derived from cancer cells, CSCs, adipocytes themselves, and surrounding microenvironments.^{41, 42} In addition, the differentiation status of adipocytes, for example, long-term adipogenic differentiation (LTAD), is the factor that regulates the secretion level of multiple adipokines from adipocytes.⁴³

Under normal physiological conditions, these adipokines perform valuable functions to maintain homeostasis. However, the dysregulation of adipokine secretion in CAA promotes CSC properties and the growth and spread of cancer cells (Figure 3).

4.1.1 Interleukin (IL)-6

Interleukin-6 is a glycosylated, 22–27-kDa secreted glycoprotein with pleiotropic effects on inflammation, immune response, and hematopoiesis and is produced in response to environmental stressors such as infections and tissue injuries.⁴⁴ IL-6 activates the Janus kinase (JAK)/STAT and mitogen-activated protein kinase (MAPK) cascades via the signal transducer glycoprotein-130 (gp-130), LIF receptor, and oncostatin M (OSM) receptor.

Interleukin-6 functions as a stimulator of various types of CSCs, such as colorectal, breast, and ovarian cancers, and activates CSC properties through its induction of OCT4, ZEB2, NANOG, and FRA1, a member of the FOS family of transcription factors through the JAK/STAT3 pathway,²⁸ and the stemness and metastasis-related genes, such as WNT5A, WNT5B, WNT7A, matrix metalloproteinase 2 (MMP-2), MMP-9, TWIST, NODAL, SDF1, and ZEB2.²⁸ Multiple copies in T-cell Malignancy 1 (MCT-1/MCTS1) and Syndecan-1 are upstream regulators of IL-6 secretion in cancer cells.^{5, 45}

In breast cancer, IL-6 secreted from adipocytes enhances mammosphere formation and CSC marker expression by activating Src. Activated Src, in turn, upregulates Sox2, and Sox2-dependent miR-302b induction increases CSC-like properties.²⁴ The combination of anti-IL-6 antibody and cisplatin destroys the lung cancer organoids, while cisplatin alone is not.⁴⁶

4.1.2 Leptin

Leptin and adiponectin are among the first adipokines discovered in the 1990s. Leptin, derived from the Greek word leptos, meaning thin, is a multifunctional neuroendocrine peptide hormone secreted from fat cells in WAT in response to the overall amount of energy stored and acute changes in energy intake. Leptin, 16 kDa in size and comprising 167 amino acids (including a 21-amino-acid secretory signal sequence), is originally identified as the product of the obese (ob) gene of the genetically obese (ob/ob) mice⁴⁷ and generally functions in appetite suppression, energy reserve, metabolism, immune response, and reproductive processes.

Leptin acts through six isoforms of transmembrane receptors called LEPRs (LEPRa through LEPRf), which are widely distributed throughout the body. LEPRs differ in the length of their intracellular domain and are structurally similar to the class I family of cytokine receptors.

Hijacking leptin and LEPR signaling is involved in the benign-to-malignant transition and functions as an inducer of cancer stemness. Leptin-induced JAK/STAT3 regulates lipid metabolism and promotes breast CSCs.⁴⁸ Tumor growth, self-renewal, and cell proliferation are significantly reduced in LEPR-silenced cells in vivo.⁴⁹ The expression of the CSC transcription factors CD44, NANOG, SOX2, and OCT4 is reduced as a result of LEPR silencing.^{49, 50} Mammary adipocyte-derived leptin upregulates STAT3-induced carnitine palmitoyltransferase 1B (CPT1B) expression and FA β-oxidation activity in breast CSCs.⁵⁰

4.1.3 Adiponectin

Adiponectin is produced almost exclusively by adipocytes in WAT and at lower concentrations in BAT. Its plasma concentration is inversely correlated with body mass index (BMI) and is inversely linked to the risk of obesity-associated malignancies and insulin resistance. Adiponectin has opposing biological functions to leptin and exerts antineoplastic effects. Lower plasma adiponectin levels are associated with the risk for various cancers, such as colorectal, breast, and endometrial cancers.⁵¹ Sphere formation ability is stimulated by leptin and is suppressed by adiponectin.⁵²

4.1.4 Adipsin

Adipsin (CFD) is predominantly secreted in significant amounts from adipocytes and is induced during adipogenesis by PPARγ, the master regulator of adipocyte biology. Adipsin is a serine protease that is indispensable for the activation of the alternative pathway of the complement system in which it cleaves complement factor B into Ba and Bb, and then a C3 convertase composed of Bb and complement component 3b (C3b) cleaves C3 into C3a and C3b. It has been reported that the adipsin/C3a pathway plays multiple roles, such as immune regulation, cell migration, insulin tolerance, adipocyte differentiation, and homing of HSCs.^53-55

We have reported that adipsin is an adipokine secreted from the adipocyte-derived stem cells isolated from the human mammary adipose tissue. Adipsin and its downstream effector HGF enhance the proliferation and CSC properties of breast cancer cells and patient-derived xenograft cells through the activation of alternative complement pathway¹⁷ (Figure 1).

4.1.5 Angiopoietin-like 4

Angiopoietin-like protein 4 (ANGPTL4) is a key regulator of lipoprotein lipase, which prevents their dimerization and subsequent activation. Modulation of ANGPTL4 has a significant impact on the body's processing and distribution of triglycerides and cholesterol.

Angiopoietin-like protein 4 secreted by adipocytes plays a vital role in the therapeutic resistance of cancers. The glycosylated ANGPTL4 protein binds to integrin α5β1 on the surface of ovarian cancer cells, thereby activating the c-myc/NF-kB pathway.⁵⁶ The overexpression of ANGPTL4 induces the enrichment of glioma stem-like cells characterized by Polycomb complex protein BMI1 and SOX2 expression and results in temozolomide resistance in glioblastoma.⁵⁷

4.1.6 Monocyte chemoattractant protein-1

Monocyte chemoattractant protein-1 (MCP-1, CCL2), the first discovered human CC chemokine located on chromosome 17 (chr.17, q11.2), is a monomeric polypeptide produced by various cell types, including human adipocytes, macrophages, and endothelial cells; and functions as a potent chemotactic factor for monocytes, memory T lymphocytes, and natural killer (NK) cells. In addition, the abundance of MCP-1 in both WAT and plasma is increased in obese mice.⁵⁸

MCP-1 protein induces Notch1 expression and enhances the CSC signaling that stimulates the sphere-forming phenotype in breast cancer cells.⁵⁹ In vitro coculture of adipocytes and breast cancer cells upregulates MCP-1 secretion from adipocytes, which in turn induces mammosphere formation and expression of CSC markers SOX2, c-MYC, and NANOG in breast cancer cells.²⁴

4.1.7 Visfatin

Visfatin, also referred to as nicotinamide phosphoribosyl transferase (NAMPT), is a 52-kDa enzyme that is involved in NAD⁺ biosynthesis.⁶⁰ The intracellular visfatin expression in cancer cells increases cell proliferation through PI3K/AKT and ERK/MAPK activation. The extracellular visfatin is secreted from several tissues with a higher secretion from adipose tissue, especially from fully differentiated WAT and BAT.

Adipokine visfatin increases stemness and angiogenesis in breast cancer. Visfatin enhances tumor sphere formation and protein expression levels of the stem cell genes NANOG, SOX2, OCT4, and Sirtuin 1 (SIRT1), which is diminished by the visfatin inhibitor FK866.⁶¹

4.1.8 Resistin

Resistin is a cysteine-rich hormone that is secreted by human peripheral blood mononuclear cells (PBMC) and macrophages and by rodent WAT and BAT. Human resistin is a peptide with a mature sequence consisting of 108 amino acids, whereas rodent resistin has 114 amino acids. The isoforms of decorin (DCN), mouse receptor tyrosine kinase-like orphan receptor 1 (ROR1), toll-like receptor 4 (TLR4), or adenylyl cyclase-associated protein 1 (CAP1) are proposed to be the resistin receptors.

Resistin has been linked to an increased risk of tumor progression in various cancer models. It is also associated with chemoresistance and stemness induction in cancer.⁶² For example, resistin promotes EMT and stemness in breast and ovarian cancer cells.⁶³ It is, however, still insufficient to conclusively link the impact of resistin on the initiation and development of cancers.

5 CONCLUSION

The incidence of obesity is rising, and greater than 40% of the world's population is expected to be overweight or suffer from obesity by 2030. Obesity increases mortality from cancers, such as prostate and stomach in men, breast (postmenopausal), cervix, and uterus in women, and kidney (renal cell), colon, esophagus (adenocarcinoma), pancreas, gallbladder, and liver in both sexes,⁵ posing obesity-related cancer as one of the leading causes of death.^{7, 79}

In this review, we discussed the molecular mechanisms of CSC regulation by adipocytes. Although adipocyte–cancer cell interactions have been extensively studied, the number of studies elucidating adipocyte–CSC interactions is still limited (Table 1). Indeed, most of the findings depend on tumor sphere assays of cancer cells, demonstrating the necessity for further studies to confirm these findings in in vivo experimental settings and using patient-derived tumor xenograft models.

Direct contact of stem cells with their niche is at least partly mediated by cell adhesion molecules, such as VCAM1/VLA4 and JAG1/NOTCH, in the hematopoietic system.⁸⁰ Similarly, it is reasonably speculated that sets of adhesion molecules will mediate the direct contact of CSCs with adipocytes inside the niche to promote CSC properties. Finally, it is reasonable to speculate that modulation of adipokine secretome and other mechanisms during LTAD will modify the ability of adipocytes to interact with CSCs. Further studies will uncover the unique roles of adipocytes as a CSC niche and the ways to overcome obesity-related cancers.

FUNDING INFORMATION

This work was supported by (1) grants-in-aid from the Japan Society for the Promotion of Science (JSPS KAKENHI) (18K07231 and 21H02769 to Y.S.; 22K15532 to B.K.); (2) the Princess Takamatsu Cancer Research Fund (to Y.S.); (3) the Fujita Health University (to B.K. and Y.S.); (4) an extramural collaborative research grant of the Cancer Research Institute, Kanazawa University (to Y.S.); (5) the Promotion and Mutual Aid Corporation for Private Schools of Japan (to Y.S.); and (6)Memorial Fund of the General Meeting of the Japanese Association of Medical Sciences (to Y.S.). The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript.

CONFLICT OF INTEREST STATEMENT

Yohei Shimono is a coinventor on a patent application owned by Stanford University (US-20110021607) describing the use of miRNAs as biomarkers for the identification and therapeutic targeting of CSCs and on a patent and a patent application owned by Fujita Health University (6664685 and 137508) describing the use of adipsin inhibitors and statins for therapeutic targeting of CSCs. Yohei Shimono holds a financial relationship with a pharmaceutical company that might be considered relevant to this study: Quanticel Pharmaceuticals, now a fully-owned subsidiary of Celgene and Bristol Myers Squibb (patent royalties). L.T. and B.K. have no conflict of interest.

ETHICS STATEMENT

DISCLOSURE

Yohei Shimono is a current Editorial Board member of Cancer Science.