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\begin{document}

\title{Aberrant pH Signaling~ Enhances Cancer Progression: The Link between pH
Regulators and Cellular~ Responses in Cancers~~~~~~~~~~~~}



\author[1]{Suzannah Miller}%
\author[2]{Yao Rong}%
\affil[1]{Department of Chemistry and Biochemistry, University of Colorado-Boulder, Boulder, Colorado, United States of America}%
\affil[2]{Department of Molecular, Cellular and Developmental Biology, University of Colorado-Boulder, Boulder, Colorado, United States of America}%


\vspace{-1em}



  
  \date{May 10, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
A reversed pH gradient is observed in nearly all types of cancer,
demonstrating that aberrant pH signaling is a hallmark of cancers. In
order to survive, both cancerous and normal cells require the ability to
sense pH changes and respond appropriately to maintain pH homeostasis.
Recent work has expanded our knowledge on the molecular basis of pH
sensing and regulation in cancers through altered expression or activity
of some ion transporters and pH-sensitive proteins (pH sensors). In this
review, we will discuss the role of aberrant pH signaling in cancers,
with a focus on how dysregulated pH dynamics affect cellular responses,
including increased cell proliferation, evasion from apoptosis,
metabolic reprogramming, directed cell migration and metastasis.
Finally, we assess advances in tools for studying pH dynamics at the
single-cell level through the development of FP-based pH biosensors
which allow us to visualize intracellular pH in live cells. Increased
understanding of the link between pH and cancer cell behaviors brings
the potential for cancer therapies via inhibition of ion transporters as
well as targeted therapeutics designed to exploit the acidic
microenvironment of tumors.%
\end{abstract}%



\sloppy


\section*{Introduction}

{\label{195516}}

Maintaining pH homeostasis inside of cells is a highly regulated and
critical process. Minor changes in pH can influence the conformation and
function of certain proteins by altering their protonation state.
Typically, internal pH (pH\textsubscript{i}) is around 7.2 with a higher
extracellular pH (pH\textsubscript{e}) around 7.4. However, cancer cells
exhibit higher pH\textsubscript{i} (7.3-7.6) and lowered
pH\textsubscript{e} (6.8-7.0), leading to a reversed pH gradient (Fig.
1a)~\hyperref[csl:1]{(White et al., 2017)}. This reversal is common across many cancer
types, making it a hallmark of carcinogenesis and an avenue for
potential therapeutic strategies~\hyperref[csl:2]{(Webb et al., 2011)}. However,
understanding dysregulation of pH in cancer is a relatively new area of
research, leaving many open questions about the role of aberrant pH
signaling in cancer cell progression.\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/Figure1-0506/Figure1-0506}
\caption{{Aberrant pH signaling in cancers\textbf{~}(a) Cancer cells have a
reversed pH gradient compared to normal cells (b) Indicated cellular
responses to a higher intracellular pH (pH\textsubscript{i}) and a lower
extracellular pH (pH\textsubscript{e}) in cancers
{\label{714588}}%
}}
\end{center}
\end{figure}

Mammalian cells maintain pH through ion transporters and pH sensors,
which make up the major pH regulators (Fig. 2). Ion transporters alter
H\textsuperscript{+} concentration while pH sensors detect and respond
to minor changes in pH. Ion transporters, such as
Na\textsuperscript{+}/H\textsuperscript{+} exchanger1 (NHE1),
H\textsuperscript{+}/lactate cotransporter (MCT), and ATP-dependent
proton pump (V-ATPase), can move H\textsuperscript{+} out of the
cell~\hyperref[csl:3]{(Parks et al., 2013)}. An increase in activity or expression of these
transporters may explain why cancers cells exhibit a more alkaline
pH\textsubscript{i} despite the increased production of acids in cancer
cells from increased metabolism and aerobic
glycolysis~\hyperref[csl:1]{(White et al., 2017}; \hyperref[csl:3]{Parks et al., 2013)}. Proteins sense variation in pH through
conformational changes facilitated by key charged residues with
protonation states that are titratable over the range of pH changes
observed between cancer and normal cells. For example, many plasma
membrane pH sensors that respond to pH\textsubscript{e} are GPCRs, such
as the ovarian cancer G-protein-coupled receptor 1 (OGR1) and G-protein
coupled receptor 4 (GPR4)~\hyperref[csl:4]{(Justus et al., 2013)}. OGR1 shows no activity at
pH 7.8 and maximum activation at pH 6.8, which has been attributed to
conformational changes in response to protonation of five pH-sensing
histidine residues~\hyperref[csl:5]{(Ludwig and Jurg, 2003)}. Histidine is often involved in pH
sensing, which may explain why somatic mutations in cancers involving
histidine occur with disproportionately high frequency, as these
mutations may provide an adaptive advantage to the high
pH\textsubscript{i} of cancers~\hyperref[csl:6]{(Kan et al., 2010)}.\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/Figure2-0506/Figure2-0506}
\caption{{The major pH regulators to maintain pH homeostasis in cancer cells: pH
transporters (ion transporters) and pH sensors (detect and respond to pH
changes)
{\label{238173}}%
}}
\end{center}
\end{figure}

The combined effects of changes in ion transporter and pH sensor
expression and activity reinforce cancer cell progression. In this
review, we summarize current understanding of how pH regulators alter pH
states in cancer and describe specific examples of proteins sensitive to
changes in pH, with a focus on how pH dynamics influence increased cell
proliferation, evasion from apoptosis, metabolic reprogramming, cell
migration, and metastasis (Fig. 1b). We also share examples of advances
in tools for monitoring pH in live cells and discuss the challenge and
potential for therapeutic applications that target pH regulators or
exploit the acidic extracellular environment around cancer cells.

\section*{Dysregulated pH promotes proliferation in
cancer}

{\label{804906}}

Since the 1980s, increased pH\textsubscript{i~}has been recognized as a
permissive signal for promoting cell proliferation. Proton
concentrations are critical for linking external stimuli to downstream
cell-cycle entry. When pH\textsubscript{i} is lower than 7.1,
mitogen-deprived fibroblasts always remain in G0/G1 quiescent state and
fail to stimulate G1 progression and cell cycle entry with external
mitogen stimuli, suggesting a potential cell-cycle dependence on
pH\textsubscript{i~}\hyperref[csl:7]{(Schreiber, 2005)}. Thus, alkaline
pH\textsubscript{i} may be required for cell-cycle entry.

Effective prevention and treatment of human cancer requires precise
mechanistic understanding of how altered pH dynamics regulate cell
proliferation. Barber et al. showed that inducing transient alkalization
at the end of S phase (DNA synthesis) could promote G2/M progression in
non-transformed normal cells, mimicking cancer cell behavior. The
activity of cyclin B/Cdk1 complex, which predominantly regulates G2/M
progression, was inhibited by blocking the transient increase in
pH\textsubscript{i}. The alkaline pH\textsubscript{i} was demonstrated
to increase cyclin B1 and Cdc25 phosphatase levels (that
dephosphorylated inhibitory Cdk1-pT15) and decrease Wee1 kinase activity
(that phosphorylates Cdk1-T15). All these findings indicate promoted
G2/M progression in cancer cells might be caused by increased amounts of
active Cdk1~\hyperref[csl:2]{(Webb et al., 2011}; \hyperref[csl:8]{Putney and Barber, 2003)}. Nevertheless, we still have a limited
understanding of how the constitutively increased cytosolic pH in cancer
cells regulates cell proliferation. One potential mechanism is suggested
by recent studies that demonstrated global histone acetylation increased
as pH\textsubscript{i} rises~\hyperref[csl:9]{(McBrian et al., 2013)}, which may induce
upregulated expression of genes required for cell cycle progression.
However, it remains unclear whether the minor increase of cytoplasmic pH
is sufficient to promote cell-cycle entry.~

Little evidence exists for how ion transporters regulate the
pH\textsubscript{i}-dependent proliferative response in distinct cancer
cells, including NHE1 and V-ATPase. NHE1, which possesses an allosteric
H\textsuperscript{+}-modifier site, has been assigned a central role in
regulating cell cycle progression~\hyperref[csl:10]{(Srivastava et al., 2008}; \hyperref[csl:11]{Stock and Pedersen, 2017)}. Efflux of
H\textsuperscript{+} from the cell by NHE1 activity in response to
mitogen stimuli leads to increased pH\textsubscript{i} and is coupled to
mTORC1 activation and downstream ribosome protein S6
phosphorylation~\hyperref[csl:12]{(Pouyss{\'{e}}gur et al., 1982)}. Disrupting NHE1 reduces the
cytosolic pH and causes cell cycle arrest both in vitro and in
vivo~\hyperref[csl:13]{(Parks et al., 2011}; \hyperref[csl:3]{Parks et al., 2013)}. Whether certain transporters are essential for
cell cycle progression across all types of cancer cells is still
understudied. However, based on current understanding, targeting
specific pH transporters might be a powerful therapeutic strategy.

\section*{Dysregulated pH promotes cell survival via evasion from
apoptosis}

{\label{714218}}

Increased pH\textsubscript{i} in cancer cells enables evasion of
chemotherapeutic cell death. Thus, over the past 30 years, scientists
have begun to exploit pHi regulation in cell death as a target for
anticancer drugs~\hyperref[csl:14]{(Shrode et al., 1997)}. Cytosolic pH is one physiological
factor considered to control the onset of apoptosis (programmed cell
death) as a decreased intracellular pH has been observed in apoptotic
cells. Notably, increased pH\textsubscript{i} in all types of cancers is
confirmed to limit apoptosis. However, the molecular basis for how cell
death evasion is modulated or triggered by changes in
pH\textsubscript{i} is mostly undetermined.

pH-mediated functional changes of apoptosis-related proteins are
important for inducing cell death. Apoptosis is initiated by release of
cytochrome c from mitochondria to cytosol, which subsequently activates
a cascade of caspases through interaction with apoptotic protease
activating factors (Apaf). The cytochrome c-mediated activation of
caspases requires cytosolic acidification, with optimum caspase activity
at pH around 6.3-6.8. This suggests that the evasion of chemotherapeutic
cell death in cancer cells is caused by blocking caspases activation at
alkaline pH\textsubscript{i}
(\textasciitilde{}7.3-7.6)~\hyperref[csl:15]{(Matsuyama et al., 2000)}. Additionally, increased
pH\textsubscript{i} causes functional change of the apoptosis activator
Bax, inhibiting apoptosis. Recent studies confirmed that increased
pH\textsubscript{i} altered the conformation of Bax, exposing
membrane-targeting domains and resulting in Bax translocation to the
mitochondrial membrane. Site-directed mutagenesis of Bax implicated
charged amino acids in the N- and C-termini as the pH-sensitive
sites~\hyperref[csl:16]{(Khaled et al., 1999)}. Collectively, constitutively increased
pH\textsubscript{i} blocks apoptotic signaling via inactivation of
caspases or inhibition of apoptotic activator Bax.

Disrupting the pH\textsubscript{i} regulatory system through the ion
transporter NHE1 restricts tumor growth, suggesting NHE1 is necessary
for pH\textsubscript{i} regulation during apoptosis through increased
pH\textsubscript{i}. An H\textsuperscript{+}-suicide technique developed
in 1984, which reverses NHE1 ion gradients, induced
H\textsuperscript{+}-mediated cell killing~\hyperref[csl:11]{(Stock and Pedersen, 2017}; \hyperref[csl:17]{Damaghi et al., 2013)}.
Similarly, acute reduction in pH\textsubscript{i} (6.0 to 5.5) is an
effective mechanism to induce cell death within minutes in
NHE1-defective cell lines~\hyperref[csl:13]{(Parks et al., 2011)}. Thus, cells lacking
pH-regulating capabilities can be killed by protons, and targeting pH
transporters to alter pH\textsubscript{i} can affect tumor cell
survival. Although the role of pH\textsubscript{i} in apoptosis is more
clear than in cell proliferation, further investigations are required to
understand whether changes in pH\textsubscript{i} are involved in other
non-apoptotic cell death pathways and which ion transporters are crucial
for pH\textsubscript{i} regulation during apoptosis.

\section*{Interconnections between pH and metabolic
reprogramming}

{\label{939318}}

Cells acquire metabolic adaptations in response to aberrant pH signaling
in cancer that support cancer's unique metabolic demands. Most tumor
cells are reprogrammed to have increased aerobic glycolysis and reduced
mitochondrial oxidative phosphorylation in response to hypoxia and
nutrient-deficient conditions (the Warburg effect). Although cancer
cells produce more lactic and carbonic acids, cells maintain an alkaline
pH\textsubscript{i} through increased expression of ion transporters
which pump protons and lactate out of the cell. Maintaining
pH\textsubscript{i} despite increased acid production from metabolism
leads to extracellular acidification but allows cells to maintain an
alkaline interior which favors increased glycolytic flux.

Glycolytic flux has been shown to increase with alkaline
pH\textsubscript{i~}\hyperref[csl:18]{(Alfarouk et al., 2014)}. However, how aberrant pH in
cancers affect metabolic reprogramming is still understudied. It's
hypothesized that the upregulated glycolytic flux at alkaline
pH\textsubscript{i} is dependent on the pH sensitivity of some
glycolytic enzymes, such as phosphofructokinase-1 (PFK-1) and lactate
dehydrogenase A (LDHA)~\hyperref[csl:1]{(White et al., 2017)}. To support this idea, prior
studies verified glycolytic flux is required for maintaining pH
homeostasis by adding 2-deoxyglucose (2-DG, glycolysis inhibitor). The
addition of 2-DG acidifies cytosol and further causes disassembly of the
V-ATPase~\hyperref[csl:19]{(Dechant et al., 2010)}. V-ATPase plays an integral role in the
tumor plasma membrane H\textsuperscript{+}-extruding system. All these
studies suggest active glycolysis is necessary for pH maintenance.

PFK-1, the first rate-limiting step in glycolysis is a well-known
pH-sensitive enzyme. The activity of PFK-1 has a more than 10-fold
increase between pH 7.0-7.4. To further study how pH dynamics affect
PFK-1 activity, Webb et al. resolved the crystal structure of the
platelet isoform of PFK-1 and identified His208 as a candidate
pH-sensing residue. Additionally, LDHA has two potential pH-sensing
domains~\hyperref[csl:2]{(Webb et al., 2011)}. However, the detailed molecular mechanisms
of pH-sensing by these glycolytic enzymes remain to be determined. In
the context of metabolism, the alkaline pH\textsubscript{i} of cancer
cells is paradoxical because increased production of lactate acids
generated by aerobic glycolysis would be predicted to lower
pH\textsubscript{i}. It could be explained by an increase in activity or
expression of pH transporters, such as MCT1/4 and
H\textsuperscript{+}-ATPase, which can pump the protons and lactate
acids out of the cells and finally causes the increased
pH\textsubscript{i~}\hyperref[csl:9]{(McBrian et al., 2013}; \hyperref[csl:3]{Parks et al., 2013}; \hyperref[csl:18]{Alfarouk et al., 2014)}.

\section*{Dysregulated pH enhances directed cell migration and
metastasis}

{\label{208302}}

Migration in both normal and cancer cells is regulated by pH, and higher
pH\textsubscript{i} and decreased pH\textsubscript{e} in cancer cells
appears to promote invasion and metastasis~\hyperref[csl:1]{(White et al., 2017)}. While
the importance of pH-regulated interactions in migration is clear and
dysregulation of pH in cancer generally enhances motility, differences
in the expression of pH regulators and sensors among different normal
and cancer cell types can lead to opposing responses to perturbations.
This section focuses on the role of NHE1 in migration while highlighting
some of the outstanding questions on differential regulation of
migration in normal cells and cancer subtypes.

In both cancerous and healthy cells, the ion transporter NHE1 is
enriched at the leading edge, creating a gradient of pH\textsubscript{i}
across the cell which is highest at the front and is critical for
migration~\hyperref[csl:20]{(Martin et al., 2010)}. In melanoma cells, examination of NHE1
using total internal reflectance fluorescence (TIRF) microscopy revealed
that NHE1 was most abundant at focal adhesions (FAs) in the front half
of the cell~\hyperref[csl:21]{(Ludwig et al., 2012)}. This led to formation of localized pH
gradients at forward FAs, where pH\textsubscript{i} was higher than the
rest of the cell and pH\textsubscript{e} was locally depressed. At FAs,
pH-sensitive proteins such as talin and focal adhesion kinase (FAK) link
actin to integrins, which span the plasma membrane and interact with the
extracellular matrix, providing points of traction. Talin binds actin
with lower affinity at higher pH, allowing faster turnover of
FAs~\hyperref[csl:10]{(Srivastava et al., 2008)}. Similarly, FAK activation through
autophosphorylation increases with higher pH\textsubscript{i}, which
facilitates FA maturation~\hyperref[csl:22]{(Choi et al., 2013)}. Higher
pH\textsubscript{i} allows dynamic assembly and disassembly of focal
adhesions at the leading edge to allow the cell to move forward.~ The
importance of NHE1 in creating a steeper pH gradient at the front of
cells is shown by loss of local pH changes at focal adhesions, increased
focal adhesion lifetimes, and slower migration in NHE1-deficient
cells~\hyperref[csl:10]{(Srivastava et al., 2008)}.~

NHE1-driven pH changes impact additional proteins which regulate
migration.~{For example, fibroblasts with a mutant NHE1 that cannot pump
protons show a loss of activation of the small GTPase Cdc42, an
important regulator of cell polarity and leading edge
protrusions~\hyperref[csl:23]{(Frantz et al., 2007)}. This effect is likely from a pH
sensitive guanine exchange factor (GEF) which promotes Cdc42 activation.
That Cdc42 is often identified as a ``master regulator'' of
polarity~\hyperref[csl:24]{(Vicente-Manzanares and Horwitz, 2011)} underscores the importance of pH in
directional cell migration.~}

NHE1 not only regulates migration through modulating pH but may itself
serve as a scaffold for proteins involved in migration. Characterization
of NHE1 expression in glioma not only showed high levels of NHE1 leading
to increased pH\textsubscript{i} but also confirmed that NHE1 directly
interacts with cytoskeletal regulators such as the actin-binding protein
ezrin~\hyperref[csl:25]{(Cong et al., 2014)}.

More work is required to define exact mechanisms for how pH enhances
migration in different cancers versus normal cells to complete our
understanding of pH dysregulation as much of the characterization of
basic mechanisms for pH-sensing in migration has been performed in
non-transformed motile cells such as fibroblasts. As NHE1 expression or
activity is upregulated in cancers, dysregulated pH is a logical
promoter for metastasis. This is supported by a comparison of wild-type
and H-ras-transformed NIH3T3 fibroblasts which showed that transformed
cells had increased export of H\textsuperscript{+} and a slightly larger
gradient of pHi from front to back, along with faster
migration~\hyperref[csl:20]{(Martin et al., 2010}; \hyperref[csl:26]{Kaplan and Boron, 1994)}.~ However, in most cases direct comparison
of cancer and normal cells or even comparison of different kinds of
cancers is challenging since expression of migration-associated proteins
may vary across cell types~\hyperref[csl:20]{(Martin et al., 2010)}. While targeting pH
regulators such as NHE1 may seem like a general strategy for blocking
invasion, inhibition of NHE1 can have opposing effects in different
cancers, leading to increased migration in some cell
types~\hyperref[csl:27]{(Lauritzen et al., 2012)}. This may be because integrin binding to the
extracellular matrix increases with decreasing pH\textsubscript{e} and
too much or too little adhesion can slow migration~\hyperref[csl:28]{(Stock et al., 2005)}.
Excess NHE1 activity may lower pH\textsubscript{e} below the optimum for
migration in some cancers, explaining why inhibition of NHE1 can
sometimes lead to increased migration. Expression of specific GPCR pH
sensors can also vary between cancer types, complicating interpretation
of their function in migration. For example, while some cancers show
elevated GPR4, Castellone et al. found that overexpression of GPR4 in
B16F10 melanoma cells promoted stress fiber formation and reduced
invasion~\hyperref[csl:29]{(Castellone et al., 2011)}. This illustrates that despite the apparent
universality of the reversed pH gradient in cancers, cell-type specific
responses need to elucidated for effective therapeutic applications.

\section*{Advanced tools to visualize pH dynamics in real time at the
single-cell
level}

{\label{710380}}

The majority of prior discoveries of pH signaling relied on measuring
cellular pH by microelectrodes and NMR. Numerous existing data suggests
that intratumoral pH\textsubscript{i} is heterogeneous, which makes
studying and interpreting pH\textsubscript{i} dynamics in model
organisms more complicated. With the rapid development of fluorescent
protein (FP)-based pH biosensors, we can now visualize intracellular pH
in each individual cell, using genetically-encoded FP-based pH
biosensors, such as pHluorin (GFP-based probe)~\hyperref[csl:30]{(Miesenb{\"{o}}ck et al., 1998)},
pHuji (RFP-based probe)~\hyperref[csl:31]{(Shen et al., 2014)}, and SRpHi (ratiometric
endosomal probe)~\hyperref[csl:32]{(Richardson et al., 2017)}. These sensors have been
underutilized in analyzing pH signaling in cancers as they were
originally developed to monitor endocytic and secretory pathways. These
sensors could potentially be used to track pH\textsubscript{i} dynamics
in cancer at the single-cell level with higher temporal and spatial
resolutions. To aid in investigating how aberrant pH signaling enhances
cancer progression, we can combine pH biosensors with other live-cell
sensors to monitor various cellular processes, such as FUCCI
sensor~\hyperref[csl:33]{(Sakaue-sawano et al., 2008)} to link pH dynamics with cell proliferation.
Thus, live-cell sensors offer a means to measure cellular pH
simultaneously with other cellular processes in real time.

FP-based biosensors have become essential tools, allowing real-time
monitoring of cellular responses within living cells. However, there are
some limitations of these pH biosensors hindering their widely
application. A number of fluorescent pH biosensors do not target a
specific pathway and randomly diffuse throughout the cytoplasm. In
addition, current pH biosensors have a narrow detectable dynamic range
(5.5-7.5)~\hyperref[csl:32]{(Richardson et al., 2017)}, making it difficult to distinguish minor
pH differences between normal and cancerous cells and relying on the
accuracy of quantitative measurements. These limitations could be
addressed by developing targeted biosensors and additional ratiometic
sensors. Overall, monitoring individual cells over time will improve our
spatiotemporal understanding of pH signaling in cancer and reveal
heterogeneity across populations of cells.

\section*{Therapeutic implications of dysregulated
pH}

{\label{668997}}

The reversed pH gradient of cancer cells poses both a challenge and an
opportunity for drug design. In general, acidic pH\textsubscript{e} at
tumors can reduce the permeability of drugs that are weak bases by
altering their charge state~\hyperref[csl:34]{(Gerweck and Vijayappa, 2006)}. On the other hand, the
acidic microenvironment may enhance the uptake of weak acids and provide
a strategy for tissue-specific delivery of drugs using nanoparticles
made of pH-sensitive polymers that release their cargo when they
encounter lower than normal physiological pH~\hyperref[csl:34]{(Gerweck and Vijayappa, 2006}; \hyperref[csl:35]{Gao et al., 2010)}.
Identification of pH regulators and sensors that promote cancer
development holds therapeutic promise. However, caution is needed in
selecting specific drug targets across different cancer types. As noted
above, specific inhibition of NHE1 by drugs such as cariporide often
reduces metastic behavior but sometimes promotes motility, requiring
cell-type specific understanding of the effects of
inhibitors~\hyperref[csl:27]{(Lauritzen et al., 2012}; \hyperref[csl:28]{Stock et al., 2005)}.

Manipulating the extracellular pH is another therapeutic avenue for
cancer treatment. For example, mice treated with oral sodium bicarbonate
showed increased pH\textsubscript{e} which reestablished a more normal
pH gradient, where pH\textsubscript{e} is greater than
pH\textsubscript{i~}\hyperref[csl:36]{(Robey et al., 2009)}. While this treatment did not
reduce primary tumor growth, it did reduce the number of metastases.
Given that the reversed pH gradient facilitates cancer cell survival and
metastasis, therapies that alter pH\textsubscript{e} and/or
pH\textsubscript{i} through strategies such as inhibition of proton
transporters could be an exciting strategy for cancer treatment.

\section*{Conclusion}

{\label{165839}}

The reversal of the pH gradient through dysregulation of ion
transporters such as NHE1 is recognized as a defining characteristic of
cancer cells. Cellular responses to aberrant pH in cancer enhance cancer
progression through increased cell proliferation and survival, metabolic
adaptation, migration, and metastasis. Increased awareness of pH as a
signaling process has led to identification of many pH sensitive
proteins in normal and cancer cells and provided a general understanding
of how increased pH\textsubscript{i} in cancer impacts cellular
responses. Although altered pH correlates with cell behaviors in
cancers, more studies are needed to address whether minor changes in pH
are sufficient to cause specific responses. While most cancers have a
reversed gradient, variation in the expression of pH transporters and
sensors can result in cell-type specific responses. Thus, future work
will need to precisely assess the role of specific proteins involved in
dysregulation of pH across different cancer types. Advances in live-cell
pH sensors is facilitating spatiotemporal understanding of pH dynamics
during cellular processes at the single-cell level. Knowledge of the
unique pH environment of cancers has already impacted treatment
strategies, and a more complete understanding of the molecular
mechanisms at play will provide new drug targets to selectively exploit
pH signaling to block tumor growth and metastasis.

~

~

~

\selectlanguage{english}
\FloatBarrier
\section*{References}\sloppy
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\phantomsection
\label{csl:29}Castellone, R.D., N.R. Leffler, L. Dong, and L.V. Yang. 2011. {Inhibition of tumor cell migration and metastasis by the proton-sensing GPR4 receptor}. \textit{Cancer Letters}. 312:197–208. doi:10.1016/j.canlet.2011.08.013.

\phantomsection
\label{csl:22}Choi, C.-hoon, B.A. Webb, M.S. Chimenti, M.P. Jacobson, and D.L. Barber. 2013. {pH sensing by FAK-His58 regulates focal adhesion remodeling}. \textit{Journal of Cell Biology}. 202:849–859. doi:10.1083/jcb.201302131.

\phantomsection
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\end{document}

