Expression and function of SLC38A5, an amino acid-coupled Na+/H+ exchanger, in triple-negative breast cancer and its relevance to macropinocytosis.

TL;DR: In this paper, the authors examined the impact of SLC38A5 on macropinocytosis in triple-negative breast cancer (TNBC) cells and found that the transport function of the amino acid transporter is coupled to induction of protein-protein interactions.

Abstract: Metabolic reprogramming in cancer cells necessitates increased amino acid uptake, which is accomplished by upregulation of specific amino acid transporters. Since amino acid transporters differ in substrate selectivity, mode of transport, and driving forces, not all tumors rely on any single amino acid transporter for this purpose. Here we report on the differential upregulation of the amino acid transporter SLC38A5 in triple-negative breast cancer (TNBC). The upregulation is evident in primary TNBC tumors, conventional TNBC cell lines, patient-derived xenograft TNBC cell lines, and a mouse model of spontaneous mammary tumor representing TNBC. The upregulation is confirmed by functional assays. SLC38A5 is an amino acid-dependent Na+/H+ exchanger which transports Na+ and amino acids into cells coupled with H+ efflux. Since the traditional Na+/H+ exchanger is an established inducer of macropinocytosis, an endocytic process for cellular uptake of bulk fluid and its components, we examined the impact of SLC38A5 on macropinocytosis in TNBC cells. We found that the transport function of SLC38A5 is coupled to induction of macropinocytosis. Surprisingly, the transport function of SLC38A5 is inhibited by amilorides, the well-known inhibitors of Na+/H+ exchanger, possibly related to the amino acid-dependent Na+/H+ exchange function of SLC38A5. The Cancer Genome Atlas database corroborates SLC38A5 upregulation in TNBC. This represents the first report on the selective expression of SLC38A5 in TNBC and its role as an inducer of macropinocytosis, thus revealing a novel, hitherto unsuspected, function for an amino acid transporter that goes beyond amino acid delivery but is still relevant to cancer cell nutrition. Summary Statement SLC38A5 is an amino acid-coupled Na+/H+ exchanger that is upregulated in triple-negative breast cancer, and its function in cancer cells goes beyond amino acid delivery; it promotes macropinocytosis, a distinct form of endocytic process for cellular uptake of proteins and other nutrients present in extracellular fluid.

Summary

Introduction

• Cancer cells reprogram their metabolism to best suit their need for accelerated proliferation associated with enhanced protein/lipid synthesis, DNA/RNA synthesis, and potentiated machinery for protection against oxidative stress and apoptosis [1-3].
• This requires increased supply of nutrients to feed into the altered metabolic pathways; amino acids represent an important group among such nutrients.
• Only recently, a couple of studies have focused on SLC38A2 in breast cancer [16,17].

Materials

• Ethylisopropyl amiloride and hexamethylene amiloride were from Sigma Aldrich (St. Louis, MO, USA).
• Amiloride, benzyl amiloride and harmaline were from Cayman Chemical (Ann Arbor, MI, USA).

Human tissues

• Human breast cancer tissues and the surrounding normal tissues were obtained from the Augusta University Tumor Bank.
• The Tumor Bank collects and maintains a repository of de-identified tumor tissues and matched normal tissues; the tumor tissue collection has the approval from the Institutional Review Board and the Human Assurance Committee.
• These tissues are available to investigators without a separate approval from the Institutional Review Board.

Animals

• The authors used three different transgenic mouse models of spontaneous breast cancer for the analysis of Slc38a5 expression: MMTV-Neu (a model for HER2-positive breast cancer), MMTV-HRAS (a model for Ras activation-associated breast cancer), and MMTV-PyMT (polyoma middle T antigen-driven breast cancer, which starts initially as estrogen receptor positive and subsequently turns into estrogen receptor-negative).
• The authors used four human non-transformed mammary epithelial cell lines and seven estrogen receptornegative breast cancer cell lines.
• MCF10A and MCF12A cells were cultured in a special medium consisting of Dulbecco’s Modified Eagle’s Medium and Ham’s F12 medium, in a 1:1 ratio, supplemented with 20 ng/ml human EGF, 0.01 mg/ml bovine insulin, 500 ng/ml hydrocortisone, and 100 ng/ml cholera toxin.
• All media contained 10% fetal bovine serum.

Uptake assays

• Uptake of [3H]-serine was used to monitor the transport function of SLC38A5.
• As there are several amino acid transporters, even for serine that is used as the substrate in the present study, that are Na+-coupled, the authors cannot specifically monitor the function of SLC38A5 by using Na+-containing uptake buffer.
• Cells were seeded in 24-well culture plates (2 x 105 cells/well) with the culture medium.
• The uptake medium (250 µl) containing [3H]-serine was added to the cells.
• On the day of the uptake assay, cells were incubated with NaCl-buffer (same as the uptake buffer except that LiCl or NMDGCl was replaced with NaCl), pH 7.5.

RT-PCR

• RNA was extracted from cells and mammary tissues from mice (normal and tumor) by TRIzol reagent (Thermo Fisher Scientific), and the RNA was reverse-transcribed using High-capacity cDNA reverse transcription kit (Thermo Fisher Scientific).
• PCR and quantitative-PCR were performed with Takara Taq Hot Start Version (TaKaRa Biotechnology, Shiga, Japan) or Power SYBR Green PCR master mix (Thermo Fisher Scientific).
• Human-specific primers were used for cell lines whereas mouse-specific primers were used for mouse tissues.
• The relative mRNA expression was determined by the 2-ΔΔCt method.
• HPRT (hypoxanthine/guanine phosphoribosyl transferase) was used as a housekeeping gene for normalization.

Macropinocytosis assay

• Cells were plated on coverslips, placed in wells in a 12 well-plate, at a density of 1x105 cells/well, and cultured with 5% CO2 at 37 oC using the culture media (with 10% fetal bovine serum) recommended for the respective cell lines.
• About 16 h prior to macropinocytosis assay, the medium was removed and fresh culture medium without the fetal bovine serum was added.
• The images represent a maximum projection intensity derived from a Z-stack.
• The fluorescence quantification was done by measuring the Corrected Total Cell fluorescence (CTCF) using Image J and the following formula: CTCF = (integrated density) - (area of cell of interest) x (mean fluorescence of background).

Statistics

• RT-PCR and uptake studies were done in triplicates and repeated twice.
• Statistical analysis was performed with a two-tailed, paired Student’s t-test for single comparison and a p value < 0.05 was considered statistically significant.
• For quantification of fluorescence signals in image analysis related to micropinocytosis and for analysis of inhibition of serine uptake by amiloride and its derivatives, ANOVA followed by Dunn’s test was used to determine the significance of difference among the different groups.

Results

• Differential expression of SLC6A14 and SLC38A5 in estrogen receptor-positive (ER+) breast cancer and triple-negative (TNBC) breast cancer Functional evidence for SLC38A5 expression in TNBC cells A unique feature that distinguishes SLC38A5 from other Na+-coupled amino acid transporters is its ability to function in the presence of Li+ in place of Na+.
• When Na+ is removed from the medium, serine uptake does not occur via SLC38A5 because its transport activity is obligatorily coupled to Na+ (or Li+) and at the same time Na+/H+ exchanger functions in the opposite direction.
• To further confirm the role of SLC38A5 as a promoter of macropinocytosis, the authors employed experimental conditions in which serine uptake occurs solely via SLC38A5.
• The authors found the expression of SLC38A5 to be significantly higher in primary breast tumor tissues than in normal breast tissue (Fig. S3A).

Discussion

• The Na+/Cl- -coupled broad-selective amino acid transporter SLC6A14 is expressed ER+ breast cancer cell lines but not in TNBC cell lines [25].
• Whenever amino acid transporters in cancer are investigated, the sole focus is on the role of these transporters in the provision of amino acids to cancer cells.
• There are about three dozen amino acid transporters in mammalian cells, and to the best of their knowledge, no amino acid transporter other than SLC38A3 and SLC38A5 possesses this interesting feature of Na+/H+ exchange.
• Even though SLC38A3 exhibits functional features that are almost identical to those of SLC38A5 [7], the expression of the former is actually decreased in breast cancer, and the decrease is independent of the hormone receptor status of the tumor (TCGA database).
• This glutamine is then released into the extracellular fluid via SLC38A3 and SLC38A5, which is then taken up by neurons via SLC38A1 and SLC38A2 for subsequent conversion into glutamate for reuse as the neurotransmitter.

Figure Legends

• Relative expression of the amino acid transporters SLC6A14, SLC38A5, and SLC1A5 in paired tissues samples of breast cancer (ER+ breast cancer and ER-negative breast cancer) and normal mammary gland.
• (B) Transport activity of SLC38A5 in the TNBC cell line MB231 as monitored by the uptake of serine, glycine, and glutamine as the substrates for the transporter.
• The transport function of SLC38A5 was monitored in an uptake buffer (pH 8.5) containing 5 mM tryptophan to suppress the involvement of SLC7A5 in the uptake and by comparing the uptake in the presence and absence of Li+.
• Mice used the study did not go through breeding or pregnancy.
• (B) Quantification of the fluorescence signals, and statistical significance among the groups.

Full text

1
Expression and function of SLC38A5, an amino acid-coupled Na
+
/H
+
exchanger, in triple-negative breast cancer and its relevance to
macropinocytosis
Sabarish RAMACHANDRAN
1
, Souad SENNOUNE
1
, Monica SHARMA
2
, Muthusamy
THANGARAJU
3
, Varshini SURESH
4
, Yangzom D. BHUTIA
1
, Kevin PRUITT
2
, and Vadivel
GANAPATHY
1,
*
1
Department of Cell Biology and Biochemistry, and
2
Department of Immunology and Molecular
Microbiology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
3
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta
University, Augusta, GA 30912, USA
4
Department of Biology, Texas Tech University, Lubbock, TX 79409, USA
Corresponding author: Vadivel Ganapathy, Ph. D., Department of Cell Biology and
Biochemistry, Texas Tech University Health Sciences Center, 3601 4
th
street, Lubbock, TX
79430, USA. Email: Vadivel.ganapathy@ttuhsc.edu
Keywords: glutamine addiction, one-carbon metabolism, intracellular alkalinization, breast
cancer, macropinocytosis, amiloride
Short Title: SLC38A5 in breast cancer and its relevance to macropinocytosis
Summary Statement
SLC38A5 is an amino acid-coupled Na
+
/H
+
exchanger that is upregulated in triple-negative breast
cancer, and its function in cancer cells goes beyond amino acid delivery; it promotes
macropinocytosis, a distinct form of endocytic process for cellular uptake of proteins and other
nutrients present in extracellular fluid.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 18, 2021. ; https://doi.org/10.1101/2021.06.17.448844doi: bioRxiv preprint

2
Abstract
Metabolic reprogramming in cancer cells necessitates increased amino acid uptake, which is
accomplished by upregulation of specific amino acid transporters. Since amino acid transporters
differ in substrate selectivity, mode of transport, and driving forces, not all tumors rely on any
single amino acid transporter for this purpose. Here we report on the differential upregulation of
the amino acid transporter SLC38A5 in triple-negative breast cancer (TNBC). The upregulation is
evident in primary TNBC tumors, conventional TNBC cell lines, patient-derived xenograft TNBC
cell lines, and a mouse model of spontaneous mammary tumor representing TNBC. The
upregulation is confirmed by functional assays. SLC38A5 is an amino acid-dependent Na
+
/H
+
exchanger which transports Na
+
and amino acids into cells coupled with H
+
efflux. Since the
traditional Na
+
/H
+
exchanger is an established inducer of macropinocytosis, an endocytic process
for cellular uptake of bulk fluid and its components, we examined the impact of SLC38A5 on
macropinocytosis in TNBC cells. We found that the transport function of SLC38A5 is coupled to
induction of macropinocytosis. Surprisingly, the transport function of SLC38A5 is inhibited by
amilorides, the well-known inhibitors of Na
+
/H
+
exchanger, possibly related to the amino acid-
dependent Na
+
/H
+
exchange function of SLC38A5. The Cancer Genome Atlas database
corroborates SLC38A5 upregulation in TNBC. This represents the first report on the selective
expression of SLC38A5 in TNBC and its role as an inducer of macropinocytosis, thus revealing a
novel, hitherto unsuspected, function for an amino acid transporter that goes beyond amino acid
delivery but is still relevant to cancer cell nutrition.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted June 18, 2021. ; https://doi.org/10.1101/2021.06.17.448844doi: bioRxiv preprint

3
Introduction
Cancer cells reprogram their metabolism to best suit their need for accelerated proliferation
associated with enhanced protein/lipid synthesis, DNA/RNA synthesis, and potentiated machinery
for protection against oxidative stress and apoptosis [1-3]. This requires increased supply of
nutrients to feed into the altered metabolic pathways; amino acids represent an important group
among such nutrients. Because of their hydrophilic nature, amino acids cannot permeate plasma
membrane by diffusion; they enter the cells via selective transporters. There are more than three
dozen amino acid transporters in mammalian cells [4]. They are grouped into seven different solute
carrier gene families, based on the similarity in primary amino acid sequence. The increased
demand for amino acids in cancer cells is met by upregulation of selective amino acid transporters.
The most studied transporters for their role in cancer include SLC1A5 (Alanine-Serine-Cysteine
Transporter 2 or ASCT2) [5-8], SLC7A5 (system L Amino acid Transporter 1 or LAT1) [5-9],
SLC7A11 (system x
-
c
transporter or xCT) [5,6,10,11], and SLC6A14 (Amino acid Transporter B
0,+
or ATB
0,+
) [5-8,12-14]. The contribution of each of these transporters to tumor promotion is the
provision of selective amino acids to cancer cells, which might impact on a multitude of
downstream metabolic pathways and signaling cascades.
SLC38 family contains several amino acid transporters [15], but there is little or no information in
the literature on the role of these transporters in cancer. Only recently, a couple of studies have
focused on SLC38A2 in breast cancer [16,17]. This transporter represents the classical Na
+
-
coupled neutral amino acid transporter known as system A, cloned and functionally characterized
in our laboratory [18]. It was called “system A” because of its preference for the amino acid
alanine. However, it transports not only alanine but also other small, hydrophilic, amino acids such
as glycine and serine. Interestingly, glutamine is an excellent substrate for this transporter.
SLC38A2 is induced by hypoxia and its expression is related to endocrine resistance in breast
cancer [16]. In triple-negative breast cancer (TNBC), the level of SLC38A2 expression is
associated with worse prognosis [17]. Both studies have implicated SLC38A2 in supplying
glutamine to cancer cells. Notwithstanding these data suggesting a role for this transporter in breast
cancer progression, its expression is actually downregulated in breast cancer, particularly in TNBC
(p<1.5x10
-8
) (TCGA database). The decreased expression in cancer is paradoxical and
counterintuitive, given the proposed role of the transporter as a tumor promoter.
In the present study, we focused on another member of the SLC38 family, namely SLC38A5. This
transporter was also cloned in our laboratory [19,20]. It is referred to as SN2 transporter, meaning
that it is the second isoform with functional features defined selectively for the amino acid
transport system N. This system is Na
+
-coupled and accepts as substrates amino acids that contain
nitrogen in the side chain (i.e., glutamine, asparagine, and histidine). However, functional
characterization of the cloned transporter revealed two interesting features: (i) it also transports
glycine, serine and methionine, with highest affinity towards serine, and (ii) it utilizes an outward-
directed H
+
gradient as a driving force in addition to an inward-directed Na
+
gradient [19,20]. The
transport mechanism involves the transfer of Na
+
and amino acid substrate in one direction coupled
to the transfer of H
+
in the opposite direction, with the overall transport process being
electroneutral. Stated differently, SLC38A5 is an amino acid-coupled Na
+
/H
+
exchanger. The
rationale for the present study to evaluate the expression and function of SLC38A5 in breast cancer
is the following: (i) SLC38A5 is a target for the oncogene c-Myc [21]; (ii) the substrate selectivity
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4
of SLC38A5 suggests a potentially a key role for this transporter in cancer-associated metabolic
pathways such as glutaminolysis (glutamine) and one-carbon metabolism (serine, glycine, and
methionine) [14]; (iii) the efflux of H
+
coupled to amino acid entry fulfills dual needs in cancer
cells, namely supply of amino acids and removal of H
+
[19,20], and (iv) Na
+
/H
+
exchanger is
known to promote macropinocytosis, a novel mechanism for cellular entry of nutrients from
extracellular medium [22,23], suggesting a possibility that SLC38A5 as an amino acid-coupled
Na
+
/H
+
exchanger might also promote macropinocytosis in cancer cells.
Materials and Methods
Materials
[2,3-
3
H]-L-Serine (specific radioactivity, >5 Ci/mmol) was purchased from Moravek, Inc. (Brea,
CA, USA). [2-
3
H]-Glycine (specific radioactivity, x Ci/mmol) and [3,4-
3
H]-glutamine (specific
radioactivity, x Ci/mmol) were purchased from Perkin Elmer Corp. (Waltham, MA, USA).
Ethylisopropyl amiloride and hexamethylene amiloride were from Sigma Aldrich (St. Louis, MO,
USA). Amiloride, benzyl amiloride and harmaline were from Cayman Chemical (Ann Arbor, MI,
USA).
Human tissues
Human breast cancer tissues and the surrounding normal tissues were obtained from the Augusta
University Tumor Bank. The Tumor Bank collects and maintains a repository of de-identified
tumor tissues and matched normal tissues; the tumor tissue collection has the approval from the
Institutional Review Board and the Human Assurance Committee. These tissues are available to
investigators without a separate approval from the Institutional Review Board.
Animals
We used three different transgenic mouse models of spontaneous breast cancer for the analysis of
Slc38a5 expression: MMTV-Neu (a model for HER2-positive breast cancer), MMTV-HRAS (a
model for Ras activation-associated breast cancer), and MMTV-PyMT (polyoma middle T
antigen-driven breast cancer, which starts initially as estrogen receptor positive and subsequently
turns into estrogen receptor-negative). These mouse lines were originally obtained from the
Jackson Laboratory (Bar Harbor, ME, USA): MMTV-Neu, stock # 002376; MMTV-HRAS, stock
# x; MMTV-PyMT, stock # 002374). Since all mice used in the present study never went through
pregnancy, we used mammary tissues from adult (~12-week-old) virgin wild type mice as a control
for comparison. These mice form spontaneous tumors in mammary glands at different ages: 8-10
months in MMTV-Neu and MMTV-HRAS mouse lines; 2-3 months in the MMTV-PyMT mouse
line). The protocol was approved by the Institutional Animal Care and Use Committee of the
Texas Tech University Health Sciences Center, Lubbock, TX, USA (IACUC approval number:
15002 for breeding protocol and 18005 for experimental protocol). All the experiments described
in this study, including the animal experiments, were conducted at this institution. At the
termination of the study, mice were killed by cervical dislocation under CO
2
anesthesia in
accordance with the guidelines from the American Veterinary Medical Association.
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5
Cell lines and culture conditions
We used four human non-transformed mammary epithelial cell lines and seven estrogen receptor-
negative breast cancer cell lines. The non-transformed cell lines are: HMEC (ATCC, Cat. no. PCS-
600-010), HBL100 (ATCC, Cat. no. HTB-124), MCF10A (ATCC, Cat. no. CRL-10317), and
MCF12A (ATCC, Cat. no. CRL10782). HMEC cells were cultured in Mammary Epithelial Cell
Basal Medium (ATCC, Cat. no. PCS-600-030) and HBL100 cells were cultured in modified
McCoy’s 5a medium (ATCC, Cat. no. 30-2007). MCF10A and MCF12A cells were cultured in a
special medium consisting of Dulbecco’s Modified Eagle’s Medium and Ham’s F12 medium, in
a 1:1 ratio, supplemented with 20 ng/ml human EGF, 0.01 mg/ml bovine insulin, 500 ng/ml
hydrocortisone, and 100 ng/ml cholera toxin. All media contained 10% fetal bovine serum. Six of
the seven estrogen receptor-negative cell lines were obtained from ATCC: BT20 (Cat. no. HTB-
19), HCC1937 (Cat. no. CRL-2336), MDA-MB231 (Cat. no. CRM-HTB-26), MDA-MB436 (Cat.
no. HTB-136), MDA-MB453 (Cat. no. HTB-131) and MDA-MB468 (Cat. no. HTB-132).
SUM1315MO2 cell line was obtained from Expasy (Cat. no. CVCL_5589). BT20 cells were
cultured in Eagle’s Minimum Essential Medium (ATCC, Cat. no. 30-2003); HCC1937 cells were
cultured in RPMI-1640 medium (ATCC, Cat. no. 30-2001); MDA-MB436 cells were cultured in
Leibovitz’s L-15 medium (ATCC, Cat. no. 30-2008), supplemented with 10 µg/ml insulin and 16
µg/ml glutathione. MDA-MB231, MDA-MB453, and MDA-MB468 cells were cultured in
Leibovitz’s L-15 medium. SUMO1315MO2 cells were cultured in Ham’s F12 medium containing
10 ng/ml EGF and 5 µg/ml insulin. All media contained 10% fetal bovine serum. All cell lines
were mycoplasma-free. Three patient-derived xenograft cell lines were provided by the TTUHSC
Cancer Center; these are identified as TXBR-100, TXBR-237 and TXBR-247. These cell lines
were cultured in the special medium described above for MCF10A.
Uptake assays
Uptake of [
3
H]-serine was used to monitor the transport function of SLC38A5. Since SLC38A5 is
a Na
+
-coupled transporter with involvement of H
+
movement in the opposite direction, the uptake
assays were done using a pH 8.5 buffer to create an outwardly directly H
+
gradient across the
plasma membrane. As there are several amino acid transporters, even for serine that is used as the
substrate in the present study, that are Na
+
-coupled, we cannot specifically monitor the function of
SLC38A5 by using Na
+
-containing uptake buffer. However, unlike other Na
+
-coupled amino acid
transporters, SLC38A5 is tolerant to Li
+
, meaning that this transporter functions when Na
+
is
replaced with Li
+
[19,20]. Therefore, we used an uptake buffer with LiCl in place of NaCl. The
composition of the uptake buffer was 25 mM Tris/Hepes, pH 8.5, containing 140 mM LiCl, 5.4
mM KCl, 1.8 mM CaCl
2
, 0.8 mM MgSO
4
, and 5 mM D-glucose. Serine is also a substrate for
SLC7A5 (LAT1), which is a Na
+
-independent transporter; therefore, uptake via this transporter
will contribute to the total uptake measured in the LiCl-containing buffer. To suppress serine
uptake that might occur via SLC7A5, the uptake buffer contained 5 mM tryptophan to compete
with and block the transport of serine mediated by SLC7A5; SLC38A5 does not transport
tryptophan and therefore SLC38A5-mediated serine uptake is not affected by tryptophan. To
determine the contribution of diffusion to the total uptake of serine, the same uptake buffer but
with LiCl replaced isosmotically with N-methyl-D-glucamine chloride (NMDGCl) was used.
Serine uptake was measured in two buffers: (i) LiCl-buffer, pH 8.5 with 5 mM tryptophan; (ii)
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The copyright holder for this preprintthis version posted June 18, 2021. ; https://doi.org/10.1101/2021.06.17.448844doi: bioRxiv preprint

Frequently asked questions

Q1. What is the effect of serine on macropinocytosis?

Since macropinocytosis is promoted by alkalinization on the cytoplasmic surface of the plasma membrane, which alters actin polymerization and hence impacts formation of macropinosomes, the decrease in the cellular uptake of the fluorescent marker for macropinocytosis is expected in a Na+-free extracellular medium. 

Q2. What is the effect of the amino acid exchange on cancer cells?

The amino acid-dependent Na+/H+ exchange activity of SLC38A5 promotes macropinocytosis, which may contribute significantly to amino acid nutrition in cancer cells. 

Q3. What are the contributions mentioned in the paper "Expression and function of slc38a5, an amino acid-coupled na+/h+ exchanger, in triple-negative breast cancer and its relevance to macropinocytosis" ?

In this paper, the authors focused on another member of the SLC38 family, namely SLC 38A5. 

Q4. What are the future works in "Expression and function of slc38a5, an amino acid-coupled na+/h+ exchanger, in triple-negative breast cancer and its relevance to macropinocytosis" ?

Tumors with high expression of SLC38A5 might be more sensitive to such drugs than tumors with low expression of SLC38A5 ; this is however only a hypothesis at present, but it is a concept that deserves further investigation in the future. 

Q5. Why do amino acids enter the cell via selective transporters?

Because of their hydrophilic nature, amino acids cannot permeate plasma membrane by diffusion; they enter the cells via selective transporters. 

Q6. What is the effect of NH4Cl on the pH of cells?

During the preincubation with NH4Cl, intracellular pH increases because of the conversion of NH3 to NH4+ inside the cells and then the pH comes down to physiological pH because of the various pH regulatory mechanisms. 

Q7. What is the role of SLC38A5 in the endocytic process?

Since macropinocytosis facilitates the cellular entry of macromolecules, including proteins, present in the extracellular fluid, SLC38A5 contributes to amino acid nutrition in TNBC by two totally independent mechanisms, firstly by the traditional delivery of amino acids via the function of SLC38A5 as an amino acid transporter, and secondly by the promotion of macropinocytosis via the function of SLC38A5 as an amino acid-dependent Na+/H+ exchanger. 

Q8. What was used for RT-PCR to monitor the expression pattern of several amino acid transporters?

RNA was used for RT-PCR to monitor the expression pattern of several amino acid transporters that might be of relevance to tumor growth. 

Q9. What amino acids are good substrates for SLC38A5?

Considering that glutamine, glycine, and cysteine are good substrates for SLC38A5, efflux of these amino acids via the transporter might result in decreased cellular levels of the antioxidant glutathione (g-glutamylcysteinyl-glycine) in advanced tumors. 

Q10. What does serine do in a Na+-free medium?

When Na+ is removed from the medium, serine uptake does not occur via SLC38A5 because its transport activity is obligatorily coupled to Na+ (or Li+) and at the same time Na+/H+ exchanger functions in the opposite direction. 

Q11. What is the effect of amiloride on macropinocytosis?

Since SLC38A5 also functions as a Na+/H+ exchanger but in an amino acid-dependent manner, the authors wondered whether the promotion of macropinocytosis caused by serine in a NaCl-containing extracellular medium was sensitive to inhibition by amiloride. 

Q12. What is the effect of the amino acid derivatives on macropinocytosis?

Amiloride and its derivatives are known inhibitors of macropinocytosis because of their ability to inhibit Na+/H+ exchanger [22, 23]. 

Q13. Why did the authors focus on the transporters for glutamine?

The authors focused on the transporters for glutamine because of its multiple biological functions in cancer cell proliferation and growth. 

Q14. What was the effect of the glutamine uptake in the presence of Li+?

Glutamine uptake and glycine uptake were also increased in the presence of Li+, but the fold-stimulation elicited by Li+ was smaller compared to what was seen with serine uptake. 

Q15. What is the role of SLC38A5 in the transport of amino acids into cancer cells?

It is possible that SLC38A5 plays a role in the transport of amino acids into cancer cells during initial stages of carcinogenesis when the extracellular pH is not acidic. 

Q16. What buffer was used to measure serine uptake?

Serine uptake was measured in two buffers: (i) LiCl-buffer, pH 8.5 with 5 mM tryptophan; (ii)6NMDGCl-buffer, pH 8.5 with 5 mM tryptophan. 

Q17. What medium was used to culture BT20 cells?

BT20 cells were cultured in Eagle’s Minimum Essential Medium (ATCC, Cat. no. 30-2003); HCC1937 cells were cultured in RPMI-1640 medium (ATCC, Cat. no. 30-2001); MDA-MB436 cells were cultured in Leibovitz’s L-15 medium (ATCC, Cat. no. 30-2008), supplemented with 10 µg/ml insulin and 16 µg/ml glutathione. 

Q18. What is the transport activity of SLC38A5 in the TNBC cell line?

(B) Transport activity of SLC38A5 in the TNBC cell line TXBR-100 as monitored by the uptake of serine, glycine, and glutamine as the substrates for the transporter. 

Q19. What was the main strategy used to alter the transmembrane H+ gradient?

For this, the authors used the NH4Cl prepulse method to cause intracellular acidification, another strategy to alter the transmembrane H+ gradient. 

Q20. What is the role of SLC38A5 in the glutamine-glutamate cycle?

In normal physiology, this potential for bidirectional function of SLC38A3 and SLC38A5 is important in the glutamine-glutamate cycle that occurs in the brain between glutamatergic neurons and the surrounding astrocytes [37, 38].