Solute carrier proteins and c-Myc: a strong connection in cancer progression

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Solute carrier proteins and c-Myc: a strong connection in cancer progression Q3

Suman Pandaz, Nilanjan Banerjeez and Subhrangsu Chatterjee

Q4 Department of Biophysics, Bose Institute, P-1/12 CIT Road, Scheme VIIM, Kankurgachi, Kolkata, 700054, India

Solute carrier proteins (SLCs), the most understudied and second largest group of membrane proteins, maintain cellular metabolic homeostasis via the export and import of various solute, ions, metabolites, and even drugs. Given the importance of SLCs in maintaining normal cellular function, dysregulation of these proteins leads to the dramatic progression of cancers in neoplastic cells. The importance of these transporters as drug targets is gradually being realized by the scientific community. In this review, we describe the role of SLCs in hallmarks of cancer, focusing mainly on the connection between oncogenes (Myc) and SLCs in breast cancer. We also discussed the role of glucose and amino acid transporters in cancer cells and how they can be manipulated to develop anticancer therapies.

Introduction Q5 All cellular entities exchange molecules with their surroundings via protein-based transporter channels embedded in cell membranes. Given that the cell membrane is a selectively permeable barrier, it prevents the entry of toxic molecules from the ever-changing surrounding and serves as a barrier against pathogenic attack by bacteria, viruses, and fungi [1]. These channels and transporters import and export all the essential molecules, including water, ions, neurotransmitters, nutrients, and drugs, needed to sustain life [2]. Thus, cells need to regulate these transport mechanisms to maintain their internal homeostasis. Transport proteins represent 10% of the coding part of the human genome [3]. They comprise solute carriers, ATP-driven pumps, ion channels, water channels, and ABC transporters, among others. Of these, membrane-bound SLCs form the largest group, with huge phylogenetic diversity, and 456 SLCs have been reported in humans thus far [4]. Dysregulation of SLCs is associated with many diseases, including cancer, type 2 diabetes mellitus, gout, chronic kidney disease, and neurodegenerative disease, thus suggesting their potential as a therapeutic target [5]. Breast cancer is one of the most prevalent forms of cancer worldwide and, therefore, here we highlight the diversity of SLCs involved in amino acid and glucose metabolism in breast cancer and their

Corresponding author: Chatterjee, S. ([email protected]) z

These authors contributed equally.

1359-6446/ã 2020 Published by Elsevier Ltd. https://doi.org/10.1016/j.drudis.2020.02.007

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relationship with cancer development, providing new understanding for the selective design of SLC-targeting drugs for new-age cancer Q6 therapeutics (Table 1).

Diversity in the solute carrier family SLC family proteins are the second largest family of membrane proteins in the human genome after G-protein-coupled receptors [6]. They are integral cell membrane proteins present in the membranes of cells and organelles and facilitate the transport of molecules through symporters and antiporters [3]. Using a family root system, the HUGO Gene Nomenclature Committee (HGNC) classified SLCs into different families based on their occurrence, function, and phenotype [7]. According to the HGNC database, 456 SLCs have been reported so far, divided into 52 families; some families are subdivided into four groups by phylogenetic analysis: a (13 SLC families), b (five SLC families), d (two SLC families), and g (two SLC families) [8,9]. By contrast, depending on their sequence homology, a Protein family (Pfam) analysis divided SLCs into four distinct families: the major facilitator superfamily (16 SLC families), the amino acid/polyamine/organo cation (APC) superfamily (11 SLC families), the drug/metabolite transporter superfamily (two SLC families), and the cation:proton antiporter/anion transporter (CPA/AT) super family (two SLC families) [6,9]. The major facilitator superfamily is one of the largest families of membrane transporters in humans and includes

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ASCT2

LAT1

xCT

ATB0,+

GLUT1

SLC1A5

SLC7A5

SLC7A11

SLC6A14

SLC2A1

Insulin independent, facilitative diffusion

All neutral amino acids; all cationic amino acids Glucose, galactose, mannose, glucosamine

Fasentin Resveratrol Genistein

PPC-1 HL-60 HL-60

[83] O-(2-fluoro ethyl)-L-tyrosine

68 mM 38 mM 10–15 mM

[80–82] [(18)F]FSPG, 18F-5-FASu

HepG2 B16F10 MCF7S MCF-7 Cystine, glutamate

Electroneutral, obligatory exchange; AA exchanged for AA Unidirectional; Na+/Cl– /AA0,+ symport

6 mM 90 mM 1 mM 23  5 mM

[78,79] [(18)F]FPhPA; [99 m]Tc-labeled diethylenetriaminepentaacetic acid (DTPA-bis)- methionine MDA-MB231 HT-29 MCF-7 Large neutral amino acids Electroneutral, obligatory exchange; AA exchanged for AA

25 mM 4.1 mMs 13.9 mM

[75–77] PC3, A375 MCF-7 A549 1–20 mM 0.145 mM 250 mM

1,25dihydroxyvitamin D,L-g-glutamyl-pnitroanilide tamoxifen JHP203 BCH (2-amino-2norbornanecarboxylic acid) Sorafenib Sulfasalazine Adriamycin a-methyl-Ltryptophan Ser, Thr, Cys, Gln, Ala Electroneutral, obligatory exchange; Na+/AA exchanged for Na+/AA

Cell lines used IC50 /Ki Inhibitors/drugs Substrates Transport mechanism Protein Gene

Therapeutically targeted SLCs and their inhibitors in cancer

TABLE 1

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many solute symports and antiports with inward or outward polarity. Proteins in the same SLC family show at least 20–25% amino acid sequence identity with each other [5]. SLC proteins work in ATP-independent fashion by either facilitated diffusion along the concentration gradient or by co-transport against the concentration gradient of another solute, regulating tissue-specific solute movement, which is associated with many diseases [10].

Connection between hallmarks of cancer and SLC family proteins: an avenue towards drug design and discovery Cancer is a collection of diseases that develop gradually through specific steps involving the reprogramming of chromosomal dynamics, the cellular architecture and metabolism, resulting in cell proliferation and malignancy. A normal cell gradually acquires these hallmarks, and slowly progresses and evolves to a neoplastic form. To combat the ever-changing cancer cell, one needs to understand these hallmarks and the process of transformation. Hanahan and Weinberg proposed six hallmarks of cancer that regulate oncogenic transformation and malignancy [11]: (i) selfsufficiency for growth signals; (i) insensitivity to antigrowth signals; (iii) evasion of apoptosis; (iv) sustained angiogenesis; (v) limitless replicative potential; and (vi) tissue invasion and metastasis. Over the past few years, two more hallmarks have emerged: escaping the immune response and deregulation of cellular metabolism and energetics [12]. Cellular metabolism is dependent on the efflux and influx of nutrients through membrane transporters such as SLCs, as well as ABC transporters. Thus, some SLCs are upregulated in tumor cells because of the increased demand for energy and nutritional needs [3]. Overexpression of SLCs can increase chemosensitivity and induce cancer cell stemness. Thus, SLCs are strongly correlated with all the hallmarks of cancer reported so far (Fig. 1), highlighting them as one of the most important next-generation cancer therapeutic targets. Impacting the activity of SLCs can have a direct effect on tumor growth and progression.

SLC transporters as key modulators in cancer cell metabolism SLCs as amino acid transporters: a blockade of protein synthesis in cancer cells Amino acids are one of the most essential nutrients needed for the survival of all types of cell, including cancer cells. All mammalian cells, whether normal or cancerous, require essential amino acids (EAAs), including lysine, threonine, leucine, histidine, valine, methionine, isoleucine, tryptophan, and phenylalanine, from external sources because they cannot be synthesized in vivo. By contrast, nonessential amino acids (NEAAs) are synthesized in all the cells. However, to facilitate excessive cell proliferation and cancer progression, cancer cells must upregulate the expression of amino acid transporters to match the increased demand for nutrients. Although amino acids mainly serve as an important building block for protein synthesis, some amino acids have other roles. For example, glycine, glutamine and aspartic acid are required for nucleotide biosynthesis, which in turn aids cancer cell proliferation. Leucine, arginine and glutamine are required to activate the mammalian target of rapamycin (mTOR) pathway (Fig. 2), which has as significant role in cancer proliferation and metastasis. Serine

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Chemosensitivity: SLC01A2, SLC01B3, SLC22A1, SLC22A2, SLCA13, SLC38A1

Inducing angiogenesis: SLC9A1, SLC2A1, SLCA3, SLC2A4

Sustaining proliferative signalling: SLC1A5, SLC7A5, SLC38A2, SLC4A7, SLCA11, SLC12A2

Malignant transformation of cancer cell

Enabling replicative immortality: SLC1A5, SLC7A5

Evading growth suppressors and deregulating Cellular Energetics SLC5A8, SLC2A1, SLC5A1, SLC6A1, SLCA14, SLC12A2

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Resisting cell death/ apoptosis: SLC39A1, SLC39A10 SLC25A, SLC25A4

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Cancer stemness: SLC25A1, SLC2A12

Activating invasion and metastasis: SLC12A7, SLC12A6, SLC16A3, SLC19A3

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FIGURE 1

The connection between solute carrier proteins (SLCs) and Hallmarks of Cancer. SLCs have a strong connection with all the hallmarks of cancer reported thus far. Hanahan and Weinberg proposed six hallmarks of cancer that regulate oncogenic transformation and malignancy: (i) self-sufficiency for growth signals; (i) insensitivity to antigrowth signals; (iii) evasion of apoptosis; (iv) sustained angiogenesis (v) limitless replicative potential; and (vi) tissue invasion and metastasis. Over the past few years, additional hallmarks have emerged, namely escaping the immune system leading towards chemosensitivity and deregulation of cellular metabolism and energetics promoting cancer cell stemness. Cellular metabolism depends on the efflux and influx of nutrients through membrane transporters, such as SLCs. Thus, some SLCs are upregulated in tumor cells because of their increased demand for energy and nutritional needs.

also serves as one-carbon source for nucleotide synthesis and DNA methylation. Hence, blocking these elevated amino acid transporters in cancer cells could starve the cells and, thus, can be considered a new avenue in cancer therapeutics. Of the many SLCs identified to date, SLC1A5, SLC3A2, SLC6A14, SLC7A5, SLC7A11, SLC15, SLC17, SLC18, SLC36, and SLC38 have been found to serve as amino acid transporters [13]. SLC1A5 (Fig. 2), known as Alanine-Serine-Cysteine Transporter 2 (ASCT2), is an obligatory Na+-coupled transporter for alanine, serine, cysteine, threonine, and glutamine [14]. In acidic pH, it shows higher affinity for glutamate [15]. Thus, it enhances SLC1A5-mediated glutamine uptake for the reprogramming of cancer cells. SLC1A5 is overexpressed in cancer cells and interacts with many oncogenes and tumor-suppressor genes, thus promoting cancer cell proliferation. SLC1A5 is a main target of the oncogene c-Myc [16], Its expression is reduced by the tumor ¨ er suppressor, Retinoblastoma (Rb) protein. In HeLa cells, Bro et al. showed that c-Myc induces SLC1A5 and SLC7A5 (also known

as L-amino Acid Transporter 1; LAT1) in cancer cells, thus establishing the functional coupling between the two transporters, which regulates the efflux of extracellular glutamine as a substrate to take up leucine, which in turn activates the mTOR pathway [17]. The mTOR pathway has crucial role in cancer cell metabolism, proliferation, and survival. Interestingly, the functional coupling between SLC1A5 and SLC7A5 is not obligatory. Studies in A549 and LS174T cell lines showed that SLC7A5 activity is not dependent on SLC1A5 [18]. However, in breast cancer, intracellular glutamine is important for the activation of the mTOR pathway. High SLC1A5 expression and associated enhanced glutamine uptake is observed in human epidermal growth factor receptor 2 (HER-2) and triple-negative basal-like breast cancer (TNBC), in which it induces cell proliferation [18]. Tamoxifen and paclitaxel were reported to suppress SLC1A5 expression, thus inhibiting glutamine uptake [19], mTOR signaling, and metastatic progression of estrogen receptor (ER)-negative breast cancer cells. In aromatase inhibitor-resistant breast cancer, c-Myc overexpression www.drugdiscoverytoday.com

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c-MYC

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NRF2, IGF

Gln

Gln

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Glutathione (antioxidant)

Oxidative stress

Na+ Cys

DNA/RNA synthesis energy production lipid synthesis (glutamine addiction)

Chemo-resistance Protein synthesis VEGF, glycolysis (cell proliferation)

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FIGURE 2

Role of amino acid transporters and c-Myc in oncometabolism. SLC1A5, SLC7A5, SLC6A14, and SLC7A11 are upregulated in breast cancer. SLC1A5, SLC7A5, and SLC7A11 show functional coupling and enhance the proliferation of cancer cells. SLC1A5 (ASCT2) is an obligatory Na+-coupled transporter for alanine, serine, cysteine, threonine and glutamine, while SLC7A5 (LAT1) is a systemic L-amino acid transporter that transports branched-chain amino acids (isoleucine, valine, and leucine) and bulky amino acids (tryptophan, tyrosine, phenylalanine, glutamine, asparagine, and methionine). Glutamine induces nucleic acid and lipid biosynthesis. SLC7A11-mediated intracellular cysteine is used for glutathione synthesis, which results in reduced oxidative stress. Cysteine also activates mammalian target of rapamycin (mTOR), thus promoting cell proliferation. c-Myc acts as a positive regulator for SLC1A5, SLC6A14, SLC7A5, and SLC7A11. Abbreviations: HER-2, human epidermal growth factor receptor 2; IGF, Insulin-like growth factor NRF2, a basic leucine zipper (bZIP) protein that regulates the expression of antioxidant proteins; Rb, retinoblastoma protein; SERM, selective estrogen receptor modulator; STAT5, signal transducer and activator of transcription 5; VEGF, vascular endothelial growth factor.

upregulates SLC1A5 expression via crosstalk between ER and the HER-2 receptor [20]. SLC7A5 (Fig. 2), is a systemic L amino acid transporter that transports branched-chain amino acids (isoleucine, valine, and leucine) and bulky amino acids (tryptophan, tyrosine, phenylalanine, glutamine, asparagine, and methionine) [21]. Other leucinepreferring transporters are LAT2, LAT3, and LAT4. SLC3A2/SLC7A5 is a Na+ and pH-independent obligatory exchanger [21,22], whereas LAT1 does not interact with either cationic or anionic amino acids. Higher expression of SLC7A5 has been reported in the breast cancer cell lines MCF-7 and MDA-MB-231. Recent studies showed that, because hypoxia has a critical role in cancer growth and progression, the hypoxia-inducible factor HIF2a upregulates SLC7A5. c-Myc binds with the promoter of SLC7A5, thus inducing its expression. The connection between SLC7A5 and transcription factors such as cMyc and HIF2 drives the cellular uptake of leucine, which in turn activates mTOR signaling and establishes the cooperation between SLC1A5 and SLC7A5. Immunohistochemical analysis and meta-analysis data revealed that SLC7A5 expression is directly associated with increased tumor size, ER negativity, progesterone receptor (PR) negativity and high nuclear grade in breast cancer, thus making it a prognostic factor for breast cancer [23]. 4

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SLC7A8, another Na+-independent, large neutral amino acid transporter (also known as LAT2), has been reported as a predictive biomarker of good response to hormonal therapy in ER-positive breast cancer [24]. The Mammostrat test is an important prediction system for breast cancer, based on a multigene immunohistochemical assay that includes SLC7A5, along with other genes such as p53, HTF9C, NDRG1, and CEACAM5 [25]. This test helps to quantify the risk of early relapse of ER-positive breast cancers treated with tamoxifen, showing poor outcomes in patients with ER-positive breast cancer [25]. In locally advanced forms of breast cancer, SLC7A5 and carcinoembryonic antigenrelated adhesion molecules (CEACAM5 and CEACAM6] show poor responses towards neoadjuvant chemotherapy [26]. SLC6A14 (also known as ATB0,+; Fig. 2) is another important transporter with unique features. It mediates the unidirectional transport of all neutral and cationic amino acid substrates along Na+ and Cl– gradients and is coupled to membrane potential. SLC6A14 is highly expressed in ER-positive breast cancers to meet the increased demand for arginine, glutamine and leucine, which are vital for cancer cell proliferation and survival. It is also found to be in a strong connection with mTOR signaling. Given that SLC6A14 is a target of miR-23a, c-Myc regulates its expression

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Glucose

the expression of HIF1a in TNBC. In breast cancer, SLC7A11 expression profiles are regulated by its positive regulator, insulin-like growth factor-1 (IGF1) but negatively regulated by STAT3-STAT5 and miR-26b. Adriamycin subdues SLC7A11 activity and induces the expression of reactive oxygen species (ROS)-induced P-glycoprotein, resulting in chemoresistance. SLC7A11 is also involved in promoting distant metastasis in a mouse model of breast cancer. Both sorafenib and sulfasalazine inhibit SLC7A11 in cancer cells by endoplasmic reticulum stress and ferroptosis [28]. Glutamine is a key player because it is the most abundant amino acid in plasma and its carbon skeleton is used in different biomolecular structures with various functions. For example, glutamine supports the proliferation and progression of cancer cells by serving as a carbon source for the tricarboxylic acid (TCA) cycle for fatty acid and citrate synthesis, and as a substrate for nucleotide and NEAA synthesis. It is also involved in glutathione synthesis to maintain the redox balance and in activating mTOR the pathway via the activation of SLC1A5 [29] and SLC7A5. Glutamine is

Glutamine

Myc controlled in lymphocyte and cancer Myc controlled in cancer Myc controlled in lymphocytes Drug Discovery Today

FIGURE 3

Role of glucose and glutamine transporters and their regulation by Myc. c-Myc acts as a positive regulator for SLC1A5. It induces both SLC1A5 and SLC7A5 (LAT1) in cancer cells, thus establishing the functional coupling between the two transporters and regulates the efflux of extracellular glutamine as a substrate to take up leucine, which in turn activates the mammalian target of rapamycin (mTOR) pathway. Glutamine is converted into citrate inside mitochondria and, via the activity of ATP citrate lyase (ACLY), citrate is converted to acetyl-CoA, which is required for fatty acid and lipid biosynthesis. c-Myc also regulates the expression of SLC6A14 by miR-23a inhibition. By contrast, c-Myc promotes the overexpression of GLUT1 and regulates lactate dehydrogenase (LDHA) and hexokinase (HK) Q1 activity, resulting in the enhancement of the glycolysis rate and glucose metabolism. It also regulates glycine synthesis via serine in mitochondria. Abbreviations: a-KG, XXXX; GLS, XXX; GLUD, XXXX; OAA, XXX; SHMT2, XXX. www.drugdiscoverytoday.com Please cite this article in press as: Panda, S. et al.

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by inhibiting miR-23a [27]. Treating ER-positive breast cancer cells in vitro with a-methyl-L-tryptophan (a-MT) (a selective blocker of SLC6A14) triggers amino acid deprivation, thus inhibiting mTOR and activating autophagy. Prolonged treatment with a-MT also shifts the cells towards apoptosis. SLC7A11 (also known as xCT), coupled with SLC3A2, serves as a Na+-independent obligatory exchanger that imports extracellular cystine (Cys-S-S-Cys), which functions as a rate-limiting amino acid for glutathione synthesis and triggers the efflux of intracellular glutamate, needed for many cellular functions. High expression profiles of SLC7A11 have been found in many forms of cancer. It is another therapeutically important transporter for the proliferation of breast cancer, mainly ER-positive breast cancer cells and TNBC. Most breast cancer cells can survive under glutamine restriction; but a subgroup of TNBC re glutamine auxotrophs, thus requiring SLC7A11-mediated import of cysteine. The Mucin 1 transmembrane C-terminal subunit (MUC1-C) binds directly to CD44v and enhances the stability of SLC7A11. Glutamate efflux mediates

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converted into glutamate by glutaminase (GLS1) inside cells. Glutamate is also a precursor for most NEAAs, including aspartic acid and alanine. Many glutamine transporters are overexpressed in many forms of breast cancer [30]. Glutaminase overexpression (GLS1) has also been found in ER-negative breast cancer. Some cMyc-overexpressed breast cancers showed glutamine addiction, thus highlighting its potential as a therapeutic target [31]. Given that c-Myc serves as a transcription factor of SLC1A5, it also triggers glutamine influx and induces SLC6A14 and glutaminase 1 (GLS1) expression by inhibiting miR-23a and miR-23a/b, respectively (Fig. 3) [31]. HER-2 positive and TNBC show elevated levels of glutamine influx and GLS1, thus highlighting glutamine as a therapeutic target for breast cancer treatment [27]. CB-839 has been reported to have GLS-inhibiting activity in TNBC cells [32]. The therapeutic efficiency of cisplatin has also been enhanced by inhibiting GLS1 with higher specificity by bis-2-(5-phenylacetamido-1,2,4- thiadiazol-2-yl) ethyl sulfide (BPTES) in TNBC cells. Nicklin et al. showed that in vitro treatment of cancer cells with 2-aminobicyclo-(2,2,1)-heptanecarboxylic acid [29] (BCH; an inhibitor of SLC7A5–SLC3A2) or L-g-glutamyl-p-nitroanilide (GPNA; an inhibitor of SLC1A5) blocked the glutamine-dependent activation of mTOR complex 1 (mTORC1) and induced autophagy. However, the main challenges with respect to the development of GLS1 inhibitors are the presence of more than one isoform of GLS and their inherent subcellular localization. These challenges re yet to be resolved and should be a prime focus for cancer drug research. Myc is a helix-loop-helix leucine zipper family transcriptional regulator that dimerizes with Max family proteins and directly binds to the CAC(G/A)TG (E box) sequence, activating transcription of its target genes [33]. Yue et al. reported that oncogenic Myc promotes SLC7A5/SLC43A1-mediated EAA and glutamate uptake. Chromatin immunoprecipitation (ChIP) analysis in P493 and BE-2C cell lines revealed that Myc (n-Myc or c-Myc) is selectively recruited to the E box regions of the SLC7A5 and SLC43A1 genes to trigger their expression, thus allowing effective import of EAAs [34]. In turn, elevated EAAs stimulate c-Myc mRNA translation, establishing a c-Myc-SLC7A5/SLC43A1 feedforward regulatory circuit. Depletion of SLC7A5/SLC43A1 not only inhibits c-Myc expression, but also downregulates cell cycle and apoptosis regulators, such as BCL2 and CYCLIN D1, thus reprogramming the metabolic profile of cancer cells and inhibiting tumor cell growth in vitro and in vivo. Thus, this feedforward circuit induces the effective intake of nutrients and maintains homeostasis to foster tumor cell growth, proliferation and survival, thus highlighting SLC7A5 and SLC43A1 transporters as new therapeutic targets for c-Myc-overexpressing cancers. The microenvironment of cancer cells can be reprogrammed through metabolic remodeling to maintain their stemness, which depends on their niche stability, hypoxia, and acidosis. Nutrients and other molecules regulate the dynamic nature of the tumor micro-environment (TME), such as the extracellular matrix (ECM). An unstable ECM leads towards abnormal behavior of cancer stem cells (CSC). Targeting the membrane transporters in CSCs has emerged as a new potential therapeutic window. Recently, the cystine/glutamate antiporter xCT, encoded by SLC7A11, become a focus of research. in vivo and in vitro experiments demonstrated that pharmacological deletion of xCT caused amino acid starvation in ER-positive breast cancer cells and suppressed their 6

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proliferation [35]. Actinomycin D has been identified as a potential antitumor agent that significantly inhibits the activity of liver CSCs without affecting normal hepatocytes; the inhibitory effect on CSCs results from the inhibition of xCT expression and CD133 synthesis. Studies of tumor metabolomics revealed the dependency of tumor cell growth on glutamine. Mani et al. reported that epithelial–mesenchymal transition (EMT) endows tumor cells with self-renewal capacity and induces CSC production [36]. Polewski et al. showed that the SLC7A11 transporter is overexpressed in glioma, where it enhances the stemness phenotype of glioma stem cells [37]. Thus, targeting SLC7A11 combined with chemotherapy drugs could reduce the likelihood of cancer resistance and recurrence, which would improve the survival rate of patients with glioblastoma. Kim et al. revealed that metformin inhibits CSCs via the glutamine metabolic pathway. Studies confirmed that ASCT2 is more highly expressed in SW620-derived CSCs and knocking out ASCT2 or inhibiting glutamine enhanced the inhibitory effect of metformin on CSCs. However, whether the overexpression of ASCT2 is closely related to prostate CSCs is yet to be determined. Recent research demonstrated that tryptophan depletion and hypoxia preserve the phenotype of CSCs by enhancing OCT4 transcription; therefore, various tryptophan derivatives could be used to inhibit CSCs and would act through the kynurenine pathway [38].

Glucose transporters: deregulating ATP synthesis One of the main hallmarks of cancer cell growth is the enhanced production of cellular metabolites essential for the generation of new biomass nd to aid nutrient signaling. For this, there is an increased need for glucose to fuel increased glycolysis. This enhanced need for glycolysis, known as Warburg effect, and glucose uptake is facilitated by the increased presence of glucose transporters (GLUT) in the plasma membrane [39]. GLUTs are 500-amino acid-long proteins with 12 transmembrane-alpha helices and a single N-linked oligosaccharide [40]. Currently, 14 members of the mammalian facilitative glucose transporter family have been identified (GLUT1–GLUT12, GLUT14, and the HMIT;H+/myo-inositol transporter). The genes belong to the solute carrier 2A family (SLC2A; incorporating SLC2A1–SLC2A14]. Among the various GLUTs, GLUT1 and GLUT4 are the most widely studied. GLUT1 is normally found in erythrocytes, placenta and endothelial cells, but is overexpressed in B cell lymphoma, head and neck, brain, breast, cervical, pancreatic, lung, ovarian, esophageal, brain, renal, lung, cutaneous, colorectal, and endometrial cancers [41]. The intricate relationship between various oncogenes and GLUT was established when it was observed that triggering of certain oncogenes, including Ras and c-Myc (Fig. 3), and Src, and transcription factors, such as HIF-1 a, prompted the overexpression of GLUTs [42]. Burstein et al. studied the expression pattern of GLUT1 in various tumors and found increased levels of this transporter in cancer cells of diverse origins. The severity of GLUT1 expression is correlated with the fact that patients with GLUT1 overexpression have poor survival compared with those without GLUT1 overexpression [43]. Cantuaria et al. found a gradual increase in the expression of GLUT1 from borderline tumors to high-grade carcinomas [44]. The risk of death from colon

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carcinoma was 2.3-times higher in patients with high GLUT1 staining (>50% of cells GLUT1 positive) compared with those with low GLUT1 staining. Increased expression of GLUT1 has also been shown to be correlated with a poor diagnosis in lung carcinoma, human breast carcinoma, oral squamous cell carcinomas, esophageal cancer, rectal carcinoma, and squamous cell carcinoma of the tongue, among others. GLUT4 is encoded by SLC2A4 and its expression is cancer specific. It is overexpressed in breast and gastric cancers [45], whereas reduction of GLUT4 expression was observed in patients with pancreatic cancer [46]. GLUT5 is mainly found in the kidneys, testes, small intestine, muscle, and adipose tissue, and is also intricately associated with tumorigenesis and overexpressed in renal cell, breast and prostate cancers [47–49]. An elevated rate of glucose uptake is typical of highly pluripotent stem cells. Detail research showed that hypoxia regulates the expression of both GLUT1 and GLUT3 in mouse embryonic stem cells (mESCs) [50,51]. Christensen et al. silenced GLUT3 with small interfering (si)RNA in a Hues-7 human (h)ESCs cell line and showed that GLUT3 is present in the membrane of hESCs, where it regulates glucose uptake and pluripotency. The authors checked the expression of OCT4 (a pluripotency marker) and concluded that GLUT3 regulates cancer cell stemness [52]. By contrast, Shibuya et al. reported that GLUT1 is crucial for the maintenance of pancreatic, ovarian and glioblastoma CSCs. Genetical knockout of GLUT1 hindered CSC self-renewal [53], an effect that was also seen on inhibiting GLUT1 pharmacologically with WZB117. Impeding GLUT1 also denied CSCs of their tumor-initiating capability [53]. in vivo, CSCs are more sensitive to GLUT1 inhibition compared with non-stem cancer cells because glycolysis increases in response to HIF-1a and HIF-2a increases by upregulating GLUT1 in CSCs [53,54]. Thus, these data support the effect and role of GLUTs in maintaining CSCs and also show that GLUTs can be modulated to be an important and potential target for cancer.

SLC transporter as drug targets Can SLCs be used as a targets for anticancer therapy? If yes, will they be effective for curing cancer? The therapeutic targeting of cancer cells is essential for the selective killing of such cells and the careful exclusion of healthy cells. The advantages of SLCs as a drug are: (i) they are on the cell surface and, thus, can accessed by drugs directly from the blood stream, rather than the drug having to cross any cellular barriers; (ii) because SLCs remain on the cell membrane, SLC protein inhibitors would not need to enter the cytoplasm, thus reducing the risk of cytotoxicity; (iii) given that SLCs transport metabolites to the fast-growing cancer cells, inhibiting SLCs for even a short period of time, would starve the cancer cells while sparing healthy cells; and (iv] SLCs (especially amino acid transporters) have deep binding pockets, which are ideal sites for the multiple binding of inhibitors. Generally, inhibitors should be similar in appearance to the appropriate ligands so that they can fit into the groove on the transporter. Typically, inhibitors might contain a large aromatic fragment, which results in a wedge effect, blocking the transition of the transporter to the occluded conformation [55]. The substrat e-like splinter requires target recognition and site specificity; Q7 the wedge fragment provides additional contacts with the transporter to increase its affinity and simultaneously blocks its

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promotion to a transition state. A recent study demonstrated that 12 out of 435 SLCs could be used as drug targets [56], increasing interest in SLCs as targets for cancer drugs. LAT1, ASCT2, and xCT are overexpressed in a variety of tumors and could be exploited as potential anticancer targets. Sulfasalazine (Fig. 4), initially developed as an anti-inflammatory drug to treat rheumatoid arthritis, was also found to impede xCT, with anticancer effects in various cancer xenograft models. in vitro, it showed a marked reduction of lymphoma cell replication by inhibiting the xCT transporter. Researchers proposed that shortterm blockage of xCT would result in cysteine starvation in the cell and, thus, relatively fast-growing lymphoma cells would die without any harm to healthy cells or the host in general [57]. JPH203 is another Lat1-specific inhibitor and reduces the viability of mouse and human T cell acute lymphoblastic leukemia (T-ALL) cells in cell culture without harming normal healthy cells [58]. This specificity might result from the increased expression of LAT1 in T-ALL cells. A first-in-human Phase I study was designed to determine the safety, maximum-tolerated dose (MTD), and recommended dose of JPH203 [59]. a-MT (Fig. 4), a tryptophan derivative, interacts with SLC6A14 as a blocker, leading to amino acid starvation, interferes with the mTORC1 signaling pathway, and induces autophagy. Previous studies showed that the plasma levels of a-MT were 8 mM in mice when the compound was administered at 2 mg/ml. Thus, this could be used as lead compound to derive more active potent blockers of SLC6A14 for use in humans [60]. The clinically approved anticancer drug sorafenib was found to inhibit xCT function in HT-1080 cells in a dose-dependent manner and to activate ferroptosis in a variety of cellular contexts [28]. Inhibition of xCT and/or glutathione depletion might be effective in combination with other therapies to selectively target specific tumor types or sensitize them to other agents. Dixon et al. showed that sorafenib promotes endoplasmic reticulum stress, glutathione depletion, and the iron-dependent accumulation of lipid ROS in cancer cells by preventing xCT from transporting cysteine. Unlike normal cells, cancer cells are more vulnerable to glucose scarcity. Thus, researchers have tried to develop drug candidates to target GLUTs. Resveratrol (3,5,4’- trihydroxystilbene or RSV; Fig. 4) is structurally similar to tyrosine kinase inhibitors and is a polyphenolic natural product that hinders glucose uptake in human leukemic cell lines U-937 and HL-60, by blocking the internal face of GLUT1 [61]. Recent studies also revealed that RSV also prevents lactate production and hinders the Akt and mTOR signaling pathways [62]. Although RSV have been reported to be an apoptosis inducer, its effect is dependent upon its dose, the metabolic state and type of cell on which it is used [63], which results from it downregulating the expression of genes associated with the Wnt pathway, such as genes encoding b-catenin, c-Myc, and cyclin D1, also related to GLUT gene regulation. RSV inhibits GLUT1 on many levels, including: direct inhibition of the protein [64]; inhibition of mRNA expression [65,66]; regulation of transcription factors, such as HIF-1a and c-Myc, which regulate GLUT1 [67]; and regulating the expression of miRNA of GLUT1. Given its attraction as a target, several other small molecules have been isolated that target GLUT1, including phloretin, cytochalasin B, flavones, and flavonoids; however, the detailed structural or functional modes of inhibition have not yet been determined. Most GLUT inhibitors www.drugdiscoverytoday.com

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(a) Sulfasalazine

(d) Fasentin

(b) Resveratrol

(e) α-methyl-L-Tryptophan

(c) Sorafenib

(f) 1,25-Dihydroxy Vitamin D Drug Discovery Today

FIGURE 4

Examples of solute carrier protein (SLC) inhibitors. (A) Sulfasalazine, inhibitor of xCT (IC50 = 90 mM in B16F10 cells); (B) Resveratrol, inhibitor of GLUT1 (IC50 = 38 mM in HL-60 cells); (C) Sorafenib, inhibitor of xCT (IC50 = 6 mM in HepG2 cells); (D) Fasentin, inhibitor of GLUT1 (IC50 = 68 mM in PPC-1 cells); (E) a-methyl-L-tryptophan, inhibitor of ATB0,+ (IC50 = 23  5 mM in MCF-7 cells); (F) 1,25-dihydroxyvitamin D, inhibitor of ASCT2 (IC5 = 0.145 mM in MCF-7 cells).

have similar structural features [68] of their central skeletons (e.g., aromatic, heteroaromatic, olefinic, etc.), which are all characterized by the presence of, mainly phenolic, peripheral hydroxyl groups,. Interestingly, thiazolidinedione derivatives, initially designed as peroxisome proliferator-activated receptor g (PPAR g) agonists, were found to block glucose entry by inhibiting GLUT1 [69]. Phloretin was also found to bind to ER [70], resulting in research to determine whether ER ligands can act as GLUT1 inhibitors [71]. Fasentin is another small molecule that was found not only to inhibit glucose transportation by GLUT, but also to resist caspase activation, which results in removing the chemoresistance of a cell [72]. Recently, it was demonstrated that GLUT4 inhibition with ritonavir resulted in cytotoxicity in breast and ovarian cancers and the researchers were able to develop GLUT4specific inhibitors using a structure-based approach [73]. This approach marks a milestone because GLUT4 and GLUT1 share 68% homology and bypassing this limitation is no mean feat. This also shows why structure-based drug design is becoming an essential part of the drug design toolbox.

Concluding remarks Breast cancer is a common malignant form of cancer and one of the leading causes of death in women worldwide. However, many improvements in disease diagnosis and selective treatments, such as monoclonal antibody and hormonal therapies, have increased the 5-year relative survival rates for patients with breast cancer. However, there are many subsets of breast cancer, such as TNBC, for which no effective treatment strategies have been discovered, other than surgery. In addition, many anticancer drugs are not selective for cancer cells, resulting in adverse effects; in addition, they are only able to expand the life span of a patient for a few months or years. Thus, research is focusing on the identification of 8

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new, effective therapeutic targets for breast cancer. Given that nutrient transporters, such as amino acid transports and GLUTs are overexpressed in cancers, they have emerged as attractive and promising targets for the design of new therapeutic drugs for cancer. SLCs are pivotal yet understudied proteins that are required for the transport of most types of nutrients for maintaining cellular homeostasis. Some amino acid transporters, including SLC1A5, SLC7A5, SLC6A14, and SLC7A11, and the glucose transporter GLUT1 could be potential targets for the prognosis and treatment of breast cancer. Given that breast cancer cells are dependent on glutamine for their survival and proliferation, SLC1A5 and SLC7A5 have emerged as potential clinical targets for the treatment of HER-2 positive breast cancer and TNBC, and even as tracers for imaging for disease diagnosis and progression. In recent years, metabolic reprogramming has been established as a fundamental hallmark of cancer cells and, hence, SLC-mediated therapeutic approaches have become a target for research. Although SLC acts as a central hub in maintaining the metabolic state of cancer cells, it has received very little attention so far, but why is this? Ce´sar-Razquin et al. reported that, out of all the gene families, SLC shows the most skewed knowledge distribution curve and has highest publication asymmetry (i.e., it is the least studied). The best studied SLCs over the past 5 years were the same as those studied a decade ago [74]. Ce´sar-Razquin et al. highlighted various reasons for this pattern: (i) unification of the nomenclature of SLCs has been undertaken recently and, thus, they lack general common features; (ii) being a membrane protein, it is difficult to isolate and purify; hence undertaking experiments with SLCs is a timeconsuming process; (iii) because of the difficulty associated with purifying SLC, there are few structural characterizations of SLC; thus, screening and structure-based drug design cannot be undertaken; (iv) because SLCs regulate metabolic homeostasis of the cell,

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and use this information to design a molecule to bind to that specific target to hinder its function. One could also tune existing molecules to increase their efficacy. Thus, in-depth studies of the molecular and structural basis of SLCs, including their regulation, tissue specificity, protein networks, and structure–function relationships, must be undertaken and applied to homology modeling followed by structure-based drug designing to generate first-in-class novel SLC-specific anticancer drugs. Q8 Q9

Acknowledgment We thank the Bose Institute, DST, and CSIR for funding.

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their knockout leads to cellular shock and their overexpression leads to cellular toxicity. In addition, their role is sometimes compensated for by other overlapping and similar transport proteins; and (v) there are only a few antibodies available against such a diverse group of proteins, which makes molecular biology and cell biology studies of them additionally challenging. Thus, although we are beginning to understand the importance of SLCs in cancer cells, research is still in its infancy. There is a need to increase our understanding of SLC–ligand interactions at the structural level so that it would then be possible to view the 3D structure of the target, determine how it interacts with the ligand

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