- Academic Editor
Altered metabolism represents a fundamental difference between cancer cells and normal cells. Cancer cells have a unique ability to reprogram their metabolism by deviating their reliance from primarily oxidative phosphorylation (OXPHOS) to glycolysis, in order to support their survival. This metabolic phenotype is referred to as the “Warburg effect” and is associated with an increase in glucose uptake, and a diversion of glycolytic intermediates to alternative pathways that support anabolic processes. These processes include synthesis of nucleic acids, lipids, and proteins, necessary for the rapidly dividing cancer cells, sustaining their growth, proliferation, and capacity for successful metastasis. Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, with the poorest patient outcome due to its high rate of metastasis. TNBC is characterized by elevated glycolysis and in certain instances, low OXPHOS. This metabolic dysregulation is linked to chemotherapeutic resistance in TNBC research models and patient samples. There is more than a single mechanism by which this metabolic switch occurs and here, we review the current knowledge of relevant molecular mechanisms involved in advanced breast cancer metabolism, focusing on TNBC. These mechanisms include the Warburg effect, glycolytic adaptations, microRNA regulation, mitochondrial involvement, mitochondrial calcium signaling, and a more recent player in metabolic regulation, JAK/STAT signaling. In addition, we explore some of the drugs and compounds targeting cancer metabolic reprogramming. Research on these mechanisms is highly promising and could ultimately offer new opportunities for the development of innovative therapies to treat advanced breast cancer characterized by dysregulated metabolism.
Breast cancer is a significant global health problem and is the second most commonly diagnosed cancer worldwide, affecting over 1.6 million women annually with a 20% mortality rate [1, 2]. Most breast cancers can be broadly categorized as invasive or non-invasive and then further categorized by molecular features such as the presence or absence of hormone receptors and growth factor proteins. Among the invasive subtypes of breast cancer, triple-negative breast cancer (TNBC) is recognized as a particularly aggressive form, representing 10–15% of all breast cancer occurrences in women. TNBC is given the name due to its three-fold deficiency in the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), which are considered the main treatment targets in breast cancer patients. While other breast cancer subtypes have the potential of targeted treatment, patients with TNBC lack targetable receptors, resulting in limited treatment options and an overall worse prognosis. TNBC patients have a significantly higher incidence of metastasis, which is still considered the predominant cause of patient mortality. Upon metastasis, the cancer becomes increasingly more difficult to treat due to tumor heterogeneity and inability for physical intervention by surgery, creating more obstacles for clinicians as the cancer becomes more complex. At the cellular level, the success of the metastasis requires the tumor cells to adapt and exploit the stressful tumor microenvironment to acquire the necessary nutrients to survive the migration process and thrive at the new site. One of these adaptations is a cancer cell’s ability to reprogram its metabolism, using a variety of bioenergetic pathways to produce metabolic intermediates, amino acids precursors, nucleotides, lipids, and sugars to be able to successfully fuel the energetic demands of cellular proliferation. For this reason, metabolic reprogramming in cancer has been thoroughly validated and is now considered a defining hallmark of cancer [3]. This review examines a select number of molecular mechanisms, within the context of two major metabolic pathways—glycolysis and oxidative phosphorylation (OXPHOS)—that play a crucial role in TNBC progression. These mechanisms include the Warburg effect, glycolytic adaptations, microRNA regulation, mitochondrial involvement, mitochondrial calcium signaling and the emerging role of JAK/STAT signaling. Potential drug targets for some of these mechanisms that may offer new therapeutic interventions are also explored.
In the 1920s, Otto Heinrich Warburg made a groundbreaking discovery: cancer
cells, even in oxygen-rich environments, preferentially metabolize glucose to
lactate bypassing the more efficient OXPHOS pathway [4, 5, 6]. This phenomenon,
suitably known as the Warburg effect (also referred to as aerobic glycolysis),
revolutionized our understanding of cancer metabolism (Fig. 1) [4]. In the
presence of oxygen (O
Schematic representing the differences observed in glucose
metabolism between normal cells and cancer cells. In normal cells, glucose that
enters the cells undergoes glycolysis to generate pyruvate, nicotinamide adenine
dinucleotide (NAD+), and a modest amount of adenosine triphosphate (ATP). When
O
The Warburg effect was initially thought to be a way for cancers to compensate
their energy production because the mitochondria were damaged or dysfunctional.
Numerous research has challenged this notion, revealing a far more complex
scenario with mitochondrial defects not always being at the forefront of this
phenotype [4, 5, 6]. Some studies have shown mutations in mitochondrial enzymes or
mitochondrial DNA as possible contributors to the Warburg effect, however,
dysregulation of signaling pathways, activation of certain oncogenes, mutations
in tumor suppressors and the tumor microenvironment (TME) appear to be the major
contributors on the metabolic reprogramming of cancer [7, 8, 9, 10]. As these intricate
mechanisms continue to be explored, it is acknowledged that the observed
metabolic phenotype differences vary not only across cancer types but by the
degree of malignancy, with more aggressive and invasive cancers generally
exhibiting elevated glycolytic fluxes [11, 12, 13, 14]. This metabolic adaptation that
favors glycolysis, leads to the production of various glucose byproducts linked
to different metabolic pathways, such as the pentose phosphate pathway (PPP), and
the synthesis of essential biomolecules including nucleotides, amino acids, and
lipids. Similar adaptations can be observed in stem cells or cells undergoing
development, because it makes nutrient acquisition more efficient for new cell
production [15]. It is proposed that the TME, a network composed of the tumor
cells, immune cells, blood vessels, the extracellular matrix, and other,
non-tumor cell types, plays a role in selecting and sustaining this altered
metabolism. Lactate generated from aerobic glycolysis creates a highly acidic TME
that supports metabolic flexibility and genomic instability [16, 17]. A
phenomenon known as the “reverse-Warburg effect” discerns the transfer of
lactate (and other metabolites) between stromal cells and cancer cells, further
contributing to cancer cells exploiting the TME for metabolic adaption [18, 19, 20].
Moreover, as tumors outgrow their blood supply’s capacity, they favor
oxygen-independent energy production, stabilizing the transcription factor
Hypoxia-Inducible Factor 1-alpha (HIF-1
Type of breast cancer | Invasiveness | Glycolysis | OXPHOS | Ref. |
HER 2+ | Moderately invasive | Medium | Medium | [25] |
(i.e., SKBR3, MDA-MB-453) | ||||
TNBC | Very invasive | Up | Down/Medium | [25, 26] |
(i.e., MDA-MB-231, Hs578T) | ||||
*Breast cancer stem cells (BCSCs) | Very invasive | Up | Up | [27, 28] |
Inflammatory breast cancer IBC | Extremely invasive | Up | Up | [29] |
TN-IBC (i.e., SUM-149) | ||||
Endocrine resistant | Invasive | Up | Down | [30] |
(i.e., MCF7 Tamoxifen Resistant) |
“Up” ”Down” and “Medium” in the glycolysis and oxidative phosphorylation (OXPHOS) columns represent the status of these metabolic pathways compared to non-invasive, estrogen receptor (ER)+ breast cancer cells as Luminal A. *Breast cancer stem cells (BCSCs) subpopulation of heterogeneous breast cancer cells demonstrating strong self-renewal and generally identified by the expression of the surface markers CD44, CD24 (Cluster of Differentiation 44 and 24) and ALDH1 (Aldehyde Dehydrogenase 1+). HER2, Human epidermal growth factor receptor 2; TNBC, Triple-negative breast cancer; BCSCs, Breast cancer stem cells; IBC, Inflammatory breast cancer; TN-IBC, Triple-negative subtype of IBC.
Glucose is a primary source of cellular energy and is a central participant in
diverse metabolic pathways. Transporters facilitate its cellular entry, leading
to glycolysis and the eventual production of pyruvate. In comparison to other
subtypes of breast cancer, TNBC displays an elevated dependency on glycolysis
[25]. This is evident through increased glucose uptake and lactate secretion,
accompanied by the upregulation of essential glycolytic enzymes and transporters,
including glucose transporters (GLUTs), hexokinases (HKs), phosphofructokinase-1
(PFK), lactate dehydrogenase (LDH), and monocarboxylate transporter (MCT), as
illustrated in Fig. 2. The “triad” of transcription factors, which have been
extensively studied and are associated with the glycolytic phenotype in cancer,
consist of HIF-1
Diagram of the key metabolic features and mechanisms influencing
the metabolic reprogramming of triple-negative breast cancer (TNBC).
Overexpression of glucose transporters (GLUTs) increases glucose amount and flux
into the cell. TNBC cells preferentially use glycolysis for fueling alternative
metabolic pathways that are necessary for building cellular biomass and continue
proliferation (dashed arrows). Nicotinamide adenine dinucleotide phosphate
(NADPH) can be generated through different pathways in the cell and two of those
are shown here-the pentose phosphate pathway (PPP) cycle and the tricarboxylic
acid (TCA) cycle. NADPH is crucial in maintaining redox homeostasis by acting as
an antioxidant to inhibit surplus reactive oxygen species (ROS) damaging the
cell. Moderate amounts of ROS are necessary for cancer cell survival and stress
adaptation. Gene regulation occurs in the cell through microRNAs (miRNAs), which are shown
here in the cytoplasm, regulating gene expression post-transcriptionally of their
target mRNA. The triad of transcription factors (TFs) are shown in the nucleus
with their relative expressions in TNBC. STAT3, a recognized transcription factor
can function canonically or non-canonically by localizing to the mitochondria.
Focusing on the diagram of the mitochondria, the Ca
GLUT proteins, encoded by solute carrier 2 (SLC2) genes, belong to the
Major Facilitator Superfamily (MFS) of membrane transport proteins and serve as
the primary glucose transporters in mammalian cells. In humans, there are
fourteen GLUT proteins (GLUTs 1-14). Notably, GLUT 1-4 have been implicated in
various malignant tumors, including TNBC [32]. A comprehensive bioinformatics
study using public databases such as ONCOMINE, Kaplan-Meier Plotter, and cBioPortal
was carried out on a cohort of breast cancer patients to explore the intricate
connection between GLUT1, GLUT3, GLUT4, and clinical prognostic factors. The
study concluded that GLUT1 is overexpressed in TNBC patients and therefore has
potential as a novel prognostic biomarker [33]. Another study indicated that the
combined mRNA expression of SLC2A1 (GLUT1) and tumor suppressor,
Retinoblastoma protein (RB1), may be significantly correlated,
suggesting that tissues with high levels of RB1 mRNA may also exhibit
relatively higher levels of SLC2A1 mRNA in TNBC [34]. In vitro
studies have demonstrated promising results with specific inhibitors of GLUT
proteins in TNBC cell lines. These include the synthetic non-metabolizable
glucose analog, 2-deoxy-D-glucose (2-DG), anti-GLUT1 monoclonal antibody, and
phytochemicals like resveratrol. These inhibitors have been associated with
decreased glucose uptake, increased apoptosis, and enhanced radiation
effectiveness [35, 36, 37, 38]. Oncogenes such as Ras and MYC can promote glucose uptake
by upregulating HIF-1
When glucose enters the cell to undergo glycolysis, the first step of glycolysis
regulates the intracellular glucose transport through glucose phosphorylation by
hexokinases (HK1-4). HK1 is the predominant isoform found in many adult
differentiated tissues, meanwhile HK2 is more expressed during development [42, 43]. In vitro and in vivo studies have shown that, compared to
HK1, HK2 is highly expressed in cancers, particularly in highly glycolytic
cancers, as HK2 can partially enhance glucose flux (Fig. 2) [42, 43, 44, 45].
Elevated HK2 mRNA levels in TNBC cells, relative to non-TNBCs, suggest
intensified glycolytic activity and extracellular acidification [46, 47]. A
recent in vivo study by Blaha and colleagues [46] uncovered a
kinase-independent function of HK2 that contributes to metastasis. HK2 knockdown
in 4T1 cells, which are a murine TNBC equivalent cell line, affected the
expression of Epithelial-Mesenchymal Transition (EMT) genes and metastasis by
reducing SNAIL protein stability, and nuclear localization via Glycogen Synthase
Kinase (GSK3
There are key enzymatic reactions in glycolysis and one of those key steps is
when the glycolytic shunt enzyme
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) catalyzes and
modulates the levels of fructose-2,6-bisphosphate (F26BP). In turn, this affects the activity of the main rate-limiting step in glycolysis: the enzyme
phosphofructokinase-1 (PFK-1) [58]. Several studies have suggested that PFKFB4 is
upregulated in various cancers, including TNBC, as part of their metabolic
adaptation. Elevated PFKFB4 expression can result in increased levels of F26BP
which, thereby, enhances the activity of PFK-1. An in vitro
study by Dai and colleagues [58] revealed a non-canonical role
of PFKFB4 of nuclear translocation in response to hypoxia, activating
HIF-1
In anaerobic glycolysis, lactate dehydrogenase (LDH) catalyzes the reversible final step, converting pyruvate into lactate. Accumulation of the metabolic byproduct lactate and the consequent acidification of the extracellular environment is frequently observed in numerous solid tumors [61]. Monocarboxylate transporters (MCTs) facilitate lactate’s transport across cell membranes (Fig. 2). In particular, TNBC exhibits an increased expression of MCT isoforms, notably MCT1 and MCT4, which are crucial for mediating lactate expulsion and the acidification of the TME. Prior in vitro and in vivo studies have demonstrated that increased lactate production within cancer cells inhibits the immune surveillance of tumors by T-cells and natural killer (NK)- lymphocytes, consequently facilitating tumor proliferation [62]. The prognostic relevance of plasma LDH levels has been investigated in several breast cancer studies which concluded that there is a correlation between high serum LDH levels and poor overall survival (OS) and progression-free survival (PFS) in breast cancer patients [61, 63]. Therefore, the notably elevated expression of LDH-A (monomer of the tetramer LDH) in breast cancer tissues could serve as an indicator of the degree of malignancy.
The Janus kinase/signal transducer and activator of transcription or
JAK/STAT signaling pathway is involved in various physiological processes,
including immune response, cell growth, differentiation, and apoptosis. This
signaling pathway is a crucial communication hub within the cell and
dysregulation of JAK/STAT plays an instrumental role in the development and
metastasis of breast cancer [64, 65]. The activation of the JAK/STAT pathway can occur through a variety of stimuli, including leptin, a growth factor hormone, and cytokines such as interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 24 (IL-24), and interferon (IFN). IFN signaling has been linked with a lower incidence of
metastases in breast cancer patients and it has been suggested that it can be
used as a biomarker to measure the response to chemotherapy in TNBC [66]. The
JAK/STAT signaling pathway is intricately modulated through the direct
interaction with C/EBP
In vitro work in the TNBC cells, MDA-MB-231, demonstrated that IL-6
increases the rate of glucose intake and lactate generation by activating STAT3.
Furthermore, overexpression of constitutively activate STAT3, was observed in
TNBC and HER2+ patient tissues [71, 72]. STAT3 is crucial in advanced cancers
because its activation can promote glycolysis by activating transcription of
HIF-1
MicroRNAs (miRNAs) are a group of short, non-coding RNAs composed of about
20–25 nucleotides and make-up anywhere from 1–5% of the human genome [81, 82].
They play an essential role in regulating gene expression at the
post-transcriptional level through interactions with the 3
There are several miRNAs involved in breast cancer, however, here, the focus
will be on miRNAs implicated in TNBC metabolism. In vitro and in
vivo studies have identified miRNAs that exhibit significant correlations
with the Warburg effect in TNBC including miR-210-3p, miR-181a, miR-767-5p, and
miR-155 [87, 88, 89]. Some of these miRNAs are associated with glycolytic metabolism,
resulting in dysregulation of glucose consumption and lactate production by
modulating glucose transporters and metabolic enzymes including HK2, PKM2, and
LDH-A [90]. Within this context, the miR-200 group stabilizes phosphoglucose
isomerase (PGI), which contributes to cell invasion and metastasis, in
vitro and miR-210-3p has a pronounced effect on promoting aerobic glycolysis in
TNBC. Mechanistically, miR-210-3p was observed to target GPD1L, a
glycerol-3-phosphate dehydrogenase 1-like enzyme, thereby preserving the
stability of HIF-1
Evidence demonstrates that miR-155 functions as an oncogenic miRNA (oncomiR) in
human breast cancers, playing a crucial role in tumor development. MiR-155
controls the expression of a number of genes involved in differentiation,
inflammation, apoptosis, and transcriptional regulation. Experimentally and
through bioinformatics, several miR-155 targets (~149) have been
identified. These targets include the tumor suppressor PTEN, Forkhead-box protein
O3a (FOXO3a), Cyclin-dependent kinase inhibitor p27 (p27), and Casein
Kinase-1
While miRNAs primarily function in the cytoplasm, some studies have suggested
the presence of a subset of microRNAs within the mitochondria. These miRNAs,
called “mitomiRs” can regulate mitochondrial homeostasis, mtDNA maintenance, and
Ca
Mitochondria, as dynamic organelles, actively participate in a multitude of vital physiological processes. Beyond their primary function of ATP production, they are involved in the regulation of essential cellular processes such as programmed cell death, generating ROS, and certain biosynthetic intermediates needed for metabolic and signaling pathways [95, 96]. Mitochondria are also able to sense and uptake cellular calcium, which is correlated with mitochondrial ATP production and regulating various metabolic enzymes, adding to their catalog of contributions on cancer metabolism [50, 97, 98, 99, 100]. The divergence of cancer cells using OXPHOS as an energy factory is a favorable adaptation that supports the allocation of structural intermediates for sustained survival, especially in advanced and invasive cancers, such as TNBC [7, 101, 102].
A study that characterized the metabolic profile of breast cancer cells,
in vitro, revealed that among their panel of cells, TNBC cells presented
elevated glycolysis and reduced OXPHOS rates compared to other types of receptor
positive breast cancer cells [103]. Another study supplemented these findings
with tumor tissues gene expression data, reporting that OXPHOS complex subunit
genes were downregulated in TNBC tumor tissue samples compared with non-TNBC
tissues [47]. A byproduct of OXPHOS is ROS and interestingly, TNBC cells display
higher levels of ROS compared with other subtypes of breast cancer [104]. Excess
mitochondrial ROS can trigger apoptotic signals in the cell, but a moderate
amount of ROS can be used as a potent signaling molecule in order to adapt to the
stress of the TME, especially when a cancer becomes more advanced [50, 98, 101, 102, 105, 106]. For instance, the transcription factor HIF-1
Advanced cancers can also avoid unwanted cell fate and maintain survival by
modulating their calcium signaling. Calcium ions (Ca
At the OMM, mitochondria uptake their calcium through VDAC, mentioned previously
for its role in glycolytic cancers and at the inner mitochondrial membrane (IMM)
through the mitochondrial calcium uniporter (MCU) [97, 113]. Several studies have
analyzed MCU mRNA expression levels in available cancer patient
databases and found that MCU expression is elevated in breast cancer as compared
with normal tissues, and certain subtypes such as invasive ductal carcinoma have
higher MCU expression when compared with ductal carcinoma [114, 115, 116]. MCU
overexpression is also correlated with poor patient prognosis and with lymph node
invasion in breast cancer patients [106, 114, 115]. Notably, when MCU expression
was compared between 180 breast cancer patients, it was found that MCU is the
most elevated in basal-like (TNBC subtype) molecular type of breast cancer [117].
Interestingly, a majority of the studies that have investigated the role of MCU
in breast cancer migration and invasion have used TNBC cell lines to address
these questions due to the aggressive nature of this cancer [49, 106, 114, 115, 117, 118, 119, 120]. One study looking at three different TNBC cell lines, MDA-MB-231,
MDA-MB-468, and BT-549, showed that siRNA silencing of MCU reduced cell migration
in all three TNBC cell lines. Furthermore, in MCU deleted MDA-MB-231 mouse xenografts, they
observed a significant reduction in tumor progression and lymph node infiltration
[106]. Additionally, the same study found that MCU silencing reduced ROS
production, HIF-1
Advanced cancer diagnoses are often met with limited treatment options. Unlike other subtypes of breast cancer, patients with TNBC pose unique challenges for practitioners due to the lack of target receptors, therefore, targeting metabolic reprogramming in this cancer has emerged as a novel avenue. Some of these novel approaches, include the combination of metabolic inhibitors with current standard treatments, chemotherapy, radiation therapy, and immunotherapy, to improve outcomes for TNBC patients. An innovative approach utilizes nanoparticles for targeted drug delivery in cancer patients, including those with TNBC, to enhance treatment effectiveness while minimizing side effects and dosage. For example, nanoparticles have been employed as dual-delivery systems for combining chemotherapy agents with metabolic inhibitors. Some groups have investigated nanoparticles to deliver paclitaxel, a chemotherapy agent, and lonidamine, a glycolytic inhibitor to cancer cells, aiming to counter the Warburg effect and reduce the highly glycolytic phenotype of certain cancers [121, 122]. Another possible avenue involves diclofenac, a non-steroidal anti-inflammatory drug which reduced cell proliferation and increased apoptosis, particularly in TNBC by uniquely targeting c-MYC and thereby reducing glucose metabolism and lactate transport [123]. Pilot clinical trials have been exploring dietary interventions such as ketogenic diets and controlled fasting as well as the use of natural compounds in conjunction with chemotherapy and radiation treatment for advanced cancers [124, 125, 126, 127]. Some of these pilot studies have suggested that a ketogenic diet alongside chemotherapy could potentially yield better results for specific breast cancer subtypes. However, it is crucial to emphasize that there is currently no conclusive evidence regarding the effectiveness of this approach, and more extensive research and large-scale clinical trials are necessary to assess both its safety and efficacy [128].
Researchers have begun investigating a way to target MCU, as a result of the
increased interest in mitochondrial Ca
TNBC is a challenging subtype of breast cancer characterized by its lack of targetable receptors and aggressive nature. This review explored some of the mechanisms behind the complexity that underlies the metabolic reprogramming of this invasive breast cancer. Metabolic reprogramming in TNBC involves an increased reliance on glycolysis, influenced by oncogenic signaling pathways and mRNA regulation by microRNAs. Mitochondria once believed to be impaired, have now been clearly demonstrated to play an integral role in the metabolic adaptations observed in TNBC. Therapeutic approaches are advancing, including the use of metabolic inhibitors, targeted nanoparticles, and innovative drugs. Compounds targeting mitochondrial calcium uptake are emerging as potential candidates for treatment. This evolving understanding of cancer metabolism offers hope for improved therapeutic strategies and approaches to ultimately improve patient outcomes. As we continue to unravel the complexities of cancer biology and metabolic pathways, we are on the edge of significant breakthroughs that could significantly enhance patient outcomes and eventually revolutionize the field of cancer care.
Conceptualization: CDLP and EM; Literature research and analysis, writing, editing and visualization: CDLP, EM, HF, ZD, JS, CF, MS; All authors contributed to editorial changes in the manuscript. All authors read and approved the final version of the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
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This research was funded by the NIH/National Institute of General Medicine Sciences (NIGMS) SC2GM139676 to CDLP.
The authors declare no conflict of interest.
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