Apart from the interaction between adipocytes and macrophages in the breast TME, the availability of estrogen also increases, especially in postmenopausal breast cancer patients. Studies have observed a high prevalence of obesity correlated with increased serum concentrations of estrogen in postmenopausal breast cancer patients
[26]. Estrogen is a sex steroid hormone that is mainly associated with hormone receptor-positive breast cancer. This hormone is primarily produced in the granulosa cells of the ovaries in premenopausal women
[26]. Under normal physiological conditions, white adipocytes are also capable of producing sex steroid hormones. As a result, in postmenopausal women, when the ovaries stop producing estrogen, white adipocytes become the main source for its biosynthesis
[15]. A total of 18 carbon steroid estrogens (e.g., estrone and estradiol) are mainly converted from 19 carbon steroids androgens (e.g., androstenedione and testosterone) in the presence of aromatase
[26][32]. The obesity-associated increased release of proinflammatory cytokines in breast TME is accompanied by an increased expression of aromatase in white adipocytes, which then converts androgens to estrogens in the adipose tissue
[26]. In addition to this, obesity-assisted hyperinsulinemia decreases the level of steroid sex hormone-binding globulin (SHBG)
[33]. This increases the bioavailability of free estrogen, which then promotes mammary tumorigenesis.
2.4. Obesity-Associated Metabolic Remodeling
One of the several benefits of breast tumor cells having adipocytes in the microenvironment is the mobilization of stored fat for compensating higher metabolic requirements
[11]. Cancer cells require constant supplies of nutrients for their metabolic growth and energy. Various in vitro and in vivo studies concluded that cancer cells offset this need by generating de novo synthetic pathways to generate fatty acids from blood-derived glucose
[34][35]. The de novo biogenesis of fatty acid (FA) and cholesterol, under normoxic conditions, takes place through the conversion of glucose to pyruvate, which feeds into the tricarboxylic acid (TCA) cycle and generates citrate
[34][36]. This mitochondrially produced citrate is converted to acetyl Co A, a substrate of fatty acid synthesis, with the help of ATP citrate lyase
[37]. One molecule of Acetyl Co A, with seven molecules of malonyl Co A, in the presence of the fatty acid synthase (FASN) and reduced equivalent NADPH, generates the initial FA palmitate via serial condensation
[35][36]. This saturated long chain fatty acid is then further modified and desaturates into complex membrane phospholipids and triglycerides (TAGs) along the way, due to the action of different enzymes
[34]. In vivo and in situ studies reported an increased expression of FASN in breast cancer cells
[38][39]. FASN-catalyzed FA biosynthesis is stimulated by a high-carbohydrate diet and is suppressed during fasting. Acetyl CoA is also the initial substrate for cholesterol synthesis following the mevalonate pathway. The sterol regulatory element-binding proteins (SREBPs), the master regulator or transcription factor controlling the expression of enzymes involved in fatty acid and cholesterol synthesis, is also overexpressed in cancer
[35][36].
However, this fatty acid can also be supplied by the TME. In a proliferative TME, a lack of oxygen due to insufficient diffusion to an area distant from vasculature, causes hypoxia
[40]. Hypoxia triggers cancer cells to compete with the resident stromal and immune cells for metabolic nutrients
[41]. Under such conditions, cancer cells use aerobic glycolysis, where glucose-derived pyruvate is converted to lactate to generate ATP instead of using TCA cycle facilitated mitochondrial oxidative phosphorylation
[41]. To compensate for the need of fatty acids, cells may switch to alternative sources, such as acetate or the glutamine pathway, to biosynthesize lipids
[35]. However, in hypoxic conditions, the synthesis of monounsaturated fatty acids is compromised due to oxygen limitations, compromising the enzymatic reactions
[42]. The unavailability of unsaturated fatty acids, in turn, raises endoplasmic reticulum stress and triggers the activation of the uncoupled protein response (UPR), due to the over-incorporation of saturated fatty acids into the ER membrane, leading to cell death
[42].
The rapid hypertrophy of white adipocytes due to excess caloric intake coupled with a lack of energy expenditure results in increased fat storage
[43]. Because free FAs are toxic at higher concentrations, they are stored in the cytosolic lipid droplets of white adipocytes in the form of triglycerides
[40]. Triglyceride synthesis and storage in adipocytes takes place through the glycerol–phosphate pathway
[9]. Free FAs, in the presence of acyl CoA synthetase, forms 2 molecules of fatty acyl CoA. This fatty acyl CoA is acylated and dephosphorylated to diacyl glycerol in a reaction with glycerol-3-phosphate
[9]. Triglycerides are synthesized when a third molecules of fatty acyl CoA is added to the glycerol backbone in the presence of diacylglycerol transferase
[9]. Moreover, in the obese condition, the expression of FASN also increases in adipose tissues, thus influencing the de novo lipogenesis of triglycerides and their storage in adipocytes
[44]. This increased synthesis of triglycerides from free FAs and their accumulation as lipid droplets, results in adipocyte hypertrophy leading to obesity.
Although the average range of lipid droplets in other cells is 0.1 to 10 μm, in adipocytes they can accumulate up to 100 μm
[40]. Due to the proximity, breast cancer cells use lipids from adipocytes as a source of energy
[43]. Adipocytes can mobilize their stored lipids for breast cancer cell metabolic requirements through neutral lipolysis and autophagy. With the help of the catabolic enzyme adipocyte triglyceride lipase (ATGL), hormone sensitive lipase (HSL), monoacylglycerol lipase (MAGL), triglycerides, or triacylglycerol (TAG) are hydrolyzed into fatty acids (
Figure 2)
[43]. ATGL converts triglycerides to diacylglycerol. Then, in the presence of HSL, diacylglycerol converts into monoacyl glycerol
[9]. In the final step, monoacylglycerol breaks down to a free FA and glycerol via the MAGL enzyme-mediated hydrolysis process
[9]. Thus, in obesity, there is an increased availability of circulating free fatty acid for breast cancer cells to offset their continuous metabolic needs.
The excess FAs present in the obese and inflamed breast tumor microenvironment benefit cancer cells in many ways. FAs derived from adipocytes are an energy source for breast cancer cells following β-oxidation
[11][45]. FAs can produce 3–4 times the amount of energy compared to glucose
[35]. Fatty acid oxidation generates NADH, FADH
2, and acetyl CoA
[40]. NADH and FADH
2 then enters the electron transport chain to generate more energy
[40].
In addition to making the microenvironment favorable for breast cancer progression, the excess consumption of dietary trans fats in obesity also plays a role in driving the breast cancer cell to a more aggressive and metastatic phenotype. Fatty acids are essential building blocks for membrane lipids, such as phospholipids, glycolipids, and cholesterol
[40]. One in situ research reported that that the increased proportion of saturated FA used in building the membranes lipids increases the chances of the survival of breast cancer cells
[46]. The greater incorporation of saturated FAs in membrane lipids facilitates breast cancer cells’ survival against oxidative stress because they are less susceptible to peroxidation
[43]. Hence, under these circumstances, the proportion of saturated and unsaturated fatty acids in the cell membrane also plays a role in cancer cell survival and progression.
2.5. Obesity Favors Metastatic Behavior in Cancer Cells
Cell migration is an important fundamental biological response conserved not only in simple unicellular organisms, such as amoebas, but also in multicellular mammals where the process controls numerous biological events, such as embryonic development, wound healing, and immune cells invasion. It is also a significant biological phenomenon in pathological conditions, such as cancer metastasis. Cell migration is a mechanical phenomenon based on the coordination of the membrane protrusion formation, contractile force generation, and cell–matrix adhesion
[47]. The complex multistep process of a tumor cell spreading from its original or primary location, by invading the basement membrane towards the blood vessel to colonize a distant location, is known as “secondary cancer” or “metastasis”
[8]. The precise coordination and integration of signals from the adjacent cells and extracellular matrix is essential for this purpose
[48]. Tumor cells of an epithelial origin break off the primary tumor mass to migrate to distance locations in metastasis. As a result, the attainment of migratory properties with less intracellular connection is needed
[49]. The conversion of epithelial cells to the mesenchymal phenotype is an early event towards this multistep process
[48]. During this transition, epithelial cells start exhibiting mesenchymal-like characteristics by losing their polarity and cell–cell adhesion, and acquiring the malignant characteristics of cell migration and invasiveness
[50].
The epithelial-to-mesenchymal transition (EMT) was found to be aberrantly expressed under pathological conditions, such as cancer
[51]. An large number of studies have shown the role of EMT in cancer metastasis
[52][53]. The genetic alteration of transcription factors is the core regulator of EMT, causing changes in the expression of effector proteins that control cell adhesion and cell polarity
[51][54][55]. Hypoxic conditions in the primary tumor mass aggravates the process through the expression of the hypoxia-inducible factor (HIF-1)
[54]. Obesity further exacerbates the cancer condition in patients, as it propels primary tumor cells towards EMT events and facilitates malignant progression
[45][56]. One of the hallmarks of EMT is the “cadherin switch”, characterized by a loss of E-cadherin and the increased expression of N-cadherin
[57]. At the transcription level, the regulation of EMT is controlled by the transcription factors SNAIL, Twist, and ZEB-1 (zinc finger E-box-binding homobox 1 protein). They bind to the promoter region of EMT-targeted genes that downregulate the gene expression encoding for E-cadherin and increase the gene expression encoding for N-cadherin
[58].
2.6. Regulatory Proteins in Obesity-Favored Signaling Pathways towards Metastasis
While genetic and epigenetic alterations typically induce cancer initiation, the progression of cancer to the advanced stage is largely influenced by the tumor microenvironment. The hyperactivation of oncogenic proteins and the deletion of tumor suppression proteins, due to genetic mutations, causes an oncogenic transformation in cells
[59]. Many of these genetic mutations encode proteins that are components or targets of receptor tyrosine kinase (RTK) proteins controlled by PI3K-Akt and Ras-ERK cell-signaling pathways, such as the epidermal growth factor receptor (EGFR), small GTPase (e.g., Ras), serine/theonine kinases (e.g., Raf and Akt), cytoplasmic tyrosine kinase (e.g., Src), lipid kinase (e.g., phosphoinositide 3-kinase, PI3K), as well as nuclear receptors (e.g., the estrogen receptor, ER)
[60]. Under normal physiological conditions, both of the pathways control numerous physiological responses—keeping cell growth and proliferation in check, controlling stress signals, such as cellular apoptosis and DNA damage in response to growth factors and cytokine signaling or ligand binding
[59]. However, genetic alterations induce a constant activation of the proteins in these pathways, even in the absence of growth factor signaling. For example, Ras-ERK and PI3K-Akt cell-signaling pathways control estrogen receptor (ER)-dependent tumor cell survival, growth, and proliferation
[59]. In the case of obesity, this already challenged cellular ecosystem is further derailed with reciprocal paracrine and juxtaparacrine interactions of non-neoplastic cells inhabiting the microenvironment
[59]. Adipocyte-secreted factors may be critical in connecting obesity and the advanced progression of breast cancer via transducing signals from dysfunctional adipocytes to the proximal breast cancer cells
[61]. Paracrine interaction of adipokines with the cell-surface receptors on breast cancer cells, followed by the sustained activation of PI3K-Akt and Ras-ERK cell-signaling pathways, influence the metastatic behavior in breast cancer cells. To date, in vitro and in vivo studies have confirmed that obese or dysfunctional white adipocytes stimulate breast cancer progression by increasing the survival, growth, proliferation, migration, and invasion of breast cancer cells
[45][56][62][63][64][65].
To initiate their movement, cells must break off their adhesion contact with other cells and the matrix and form a protrusion in the direction of the movement. At the same time, the forward protrusion contacts the matrix as the rear portion of the cells detaches from the matrix through actomyosin contraction, pushing the cytoplasm forward in the direction of movement. The events that instigate this biological phenomenon are actin polymerization, actomyosin contraction, and cell matrix adhesion
[66][67]. The Rho family of GTPase proteins (RhoA, Rac1, and Cdc42) controls these fundamental physiological processes by activating the downstream effector proteins of multiple cells-signaling pathways, including actin regulators, adapter proteins, protein kinases, and phospholipases
[68][69]. By controlling those proteins, Rho family GTPase proteins influence normal cellular functions, such as cell adhesion, migration, and invasion
[70]. The activation of Rho family GTPase proteins is controlled by the guanine nucleotide exchange factor (GEF)
[71]. The molecular switch between the GTP and GDP bound form of Rho GTPase is catalyzed by GEFs, which displaces GDP and allows the binding of GTP on the active site of Rho family GTPase
[70]. GTP binding then ensures conformational changes in small GTPases, which enables them to interact with downstream effector proteins
[70]. The intracellular signal transducer proteins, Src and PI3K, work upstream of GEF and control its activation in response to the activation of RTK receptors in the presence of growth factors, cytokines and chemokines
[72]. Cytokine receptors of the immunoglobulin (Ig) superfamily and GPCR-type receptor can also activate GEFs in response to extracellular stimuli, and can coordinate with the Rho family GTPases’ activation signaling
[73].
While GEFs activate the small GTPases, there are two other regulatory proteins that direct their inactivation. GAP (GTPase-activating protein) is one of the regulatory proteins that induces the inactivation of small GTPases by hydrolyzing the bound GTP and switching their confirmation to the previous GDP-bound state
[70]. Guanine nucleotide dissociation inhibitors (GDIs) are another set of regulatory proteins that inactivate small GTPase proteins by sequestering them in the cytoplasm and inhibiting their localization in the plasma membrane, mediated by masking their C-terminal lipid moieties
[70]. Hence, any disruption to this regulatory activity increases the progression of cancer. Rho family GTPase proteins were observed to be overexpressed in cancer, including breast cancer
[70][74]. Their overexpression in cancer implies that there is a constant activation of GEFs in comparison to GAPs and GDIs. This further confirms the importance of the detrimental effect of the obese tumor microenvironment in the progression of cancer.