Targeting pH regulating proteins for cancer therapy–Progress and limitations

Accepted Manuscript Title: Targeting pH regulating proteins for cancer therapy − Progress and Limitations Authors: Scott K. Parks, Jacques Pouyss´egur PII: DOI: Reference:

S1044-579X(17)30007-X http://dx.doi.org/doi:10.1016/j.semcancer.2017.01.007 YSCBI 1289

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Seminars in Cancer Biology

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17-1-2017 24-1-2017

Please cite this article as: Parks Scott K, Pouyss´egur Jacques.Targeting pH regulating proteins for cancer therapy − Progress and Limitations.Seminars in Cancer Biology http://dx.doi.org/10.1016/j.semcancer.2017.01.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Seminars in Cancer Biology special issue: „The new pH-centric paradigm in oncology and medicine‟

Title: Targeting pH regulating proteins for cancer therapy – Progress and Limitations

Authors: Scott K. Parks1 and Jacques Pouysségur1,2

Affiliations: 1

Medical Biology Department, Centre Scientifique de Monaco (CSM), Monaco.

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Institute for Research on Cancer & Aging (IRCAN), CNRS, INSERM, Centre A.

Lacassagne, University of Nice-Sophia Antipolis, Nice, France

*Corresponding author contact information: [email protected] Centre Scientifique de Monaco, 8, quai Antoine 1er, MC-98000 MONACO

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Abstract Tumour acidity induced by metabolic alterations and incomplete vascularisation sets cancer cells apart from normal cellular physiology. This distinguishing tumour characteristic has been an area of intense study, as cellular pH (pHi) disturbances disrupt protein function and therefore multiple cellular processes. Tumour cells effectively utilise pHi regulating machinery present in normal cells with enhancements provided by additional oncogenic or hypoxia induced protein modifications. This overall improvement of pH regulation enables maintenance of an alkaline pHi in the continued presence of external acidification (pHe). Considerable experimentation has revealed targets that successfully disrupt tumour pHi regulation in efforts to develop novel means to weaken or kill tumour cells. However, redundancy in these pH-regulating proteins, which include Na+/H+ exchangers (NHEs), carbonic anhydrases (CAs), Na+/HCO3- co-transporters (NBCs) and monocarboxylate transporters (MCTs) has prevented effective disruption of tumour pHi when individual protein targeting is performed. Here we synthesise recent advances in understanding both normoxic and hypoxic pH regulating mechanisms in tumour cells with an ultimate focus on the disruption of tumour growth, survival and metastasis. Interactions between tumour acidity and other cell types are also proving to be important in understanding therapeutic applications such as immune therapy. Promising therapeutic developments regarding pH manipulation along with current limitations are highlighted to provide a framework for future research directives.

Keywords: pH regulation, tumour cell metabolism, hypoxia, anti-cancer strategies, pH manipulation and immune therapy

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Introduction Solid tumour cells are incredibly resistant to stressors that exist in their environment. One outstanding capability is the tolerance of external acidosis made possible via efficient pH regulatory systems. Typically in normal human physiology cells do not experience prolonged pH disturbances whereas in the tumour microenvironment increased acidity poses a considerable cellular stress. Yet this stress is not only overcome by tumour cells but also utilised to promote expansion and invasion. Consequently, it has been of great interest in the field of oncology to better understand the pH-regulating mechanism of tumour cells in hopes of developing novel targets to exploit in their physiologically altered environment. Why do cancer cells face acidic stress and how do they continue to thrive? This question has been a focal point since the pioneering work of Otto Warburg demonstrated the pre-dominance of fermentative glycolysis in cancer tissues [1-4]. At the root of tumour acidosis is the hallmark cancer feature of an abnormal/chaotic vasculature, which results in poor blood delivery and fluid clearance [5]. Thus, accumulation of lactic acid (metabolic end-product of glycolysis) [6] and conversion of CO2 (metabolic end-product of cellular respiration) [7-9] in the extracellular tumour compartment results in a substantial acidosis (Figure 1). Tumour acidity has been consistently observed in vivo and can reach values approaching pH 6 [10, 11]. Normally, when cells are exposed to metabolic acidosis, intracellular pH (pHi) values decrease [12, 13]. However, tumour cells commonly demonstrate an alkaline pHi which is favourable for cellular metabolism and proliferation (for review see [4]). Thus, alterations in tumour pH dynamics compared to normal tissues present two fundamental differences to normal physiology: (i) prolonged extracellular acidification (pHe), which contributes to a number of aspects including metastasis [14, 15], and (ii)

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increased pHi, which favours cell proliferation [16]. Therefore, the study of acid-base disturbances in tumours has to consider the distinct differences presented by the intra- and extra- cellular compartments (pHi vs. pHe). Intricate networks of membrane proteins and cytoplasmic buffers regulate pH i [17] (Figure 1). Tumour cells possess this fundamental machinery with additional enhancements provided by hypoxia regulated (primarily HIF-regulated) proteins (for recent review see [18]). As hypoxia provides a clear advantage for tumour cells during acidosis [12], the role of hypoxia-regulated proteins has been an important research topic during the past 15 years. Here we focus on recent developments in the understanding of pH regulating proteins that are providing exciting potential for novel therapeutic development. We also elaborate on how targeting of pH regulation is being considered as an adjunct strategy with other areas of oncology including immunotherapy and cancer stem cell disruption. To further focus the scope of this review, we discuss pH-regulating proteins that fall in two broad contexts: those involving transport of protons (H+/acid) and those involving transport of bicarbonate (HCO3-/base) (Figure 1). Topics that have progressed with promise for immediate clinical application are highlighted along with other concepts that are hampered by the complex redundancy in pH regulating targets.

1. H+ Transporting Proteins (i) Na+/H+ exchangers (NHEs) NHEs are the most common H+ extruding mechanisms found on cell membranes with a near ubiquitous expression of the NHE1 isoform and complementary expression of other NHE isoforms depending on tissue origin [19, 20] (Figure 2). NHE1 has been studied extensively owing to its integral role in pHi

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regulation, in particular due to its allosteric H+-activation site that is exposed to cytoplasmic changes in H+ [21-24]. Generation of NHE1-null mutant fibroblast cells provided the first strong proof that cellular acidification due to disruption of pH i regulating proteins could substantially reduce cell viability [25-29]. However, the degree of acidity required for „H+-suicide‟ is quite extreme and we have previously described limitations for achieving such a level of pHi acidification in vivo [4]. Although NHE1 inhibitors never progressed to the clinic for cancer treatment (see [4] for further discussion), these essential pHi regulating transporters remain of interest within oncology and recent work has highlighted their role in a number of vital tumour cell processes. In the past 5 years the pHi regulating role of NHE1 has been reinforced for many important aspects of cellular physiology including cell migration and invasion [30-32], proliferation [33], metastases [34] and interactions with chemotherapy [35]. Advances in genomic editing (Zinc Finger Nuclease (ZFN) and CRISPR-cas9) have further stimulated research with an ability to avoid confounding variables of NHE pharmacology. NHE1-knockout (NHE1-ko) in triple-negative breast cancer cells (MDA-MB-231) resulted in decreased migration/invasion in vitro, decreased engraftment and growth of tumour xenografts and increased sensitivity to the chemotherapeutic agent paclitaxel [36]. These NHE1-ko cells have been further used in combination with NHE1 mutations to reveal the critical role of NHE1 in metastasis and the epithelial-mesenchymal transition (EMT) via control of vimentin expression patterns [37]. NHE1-ko was further performed to assess its impact on threedimensional (3D) spheroid growth (MCF-7 and MDA-MB-231 cells) [38]. NHE1-ko (and knockdown) reduced 3D growth in both cell types with obvious morphological differences in spheroid characteristics [38]. Although NHE1 inhibition did not mimic

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effects observed with NHE1-ko in spheroids [38], these above data suggest that NHE1 indeed play an important role in numerous aspects of breast tumour cell progression. We have recently extended NHE1-ko studies to colon cancer cells [39]. Consistent with what has been observed in breast cancer cells, NHE1-ko reduced cell proliferation, pHi regulation and growth of tumour xenografts. Interestingly, we did not observe a clear synergy for combined knockout of NHE1 and carbonic anhydrase 9 (see below for extended CA description), which in theory would collapse the predominant pHi regulatory machinery. Furthermore, circumstantial evidence has suggested that NHE1 may be more important in normoxic as compared to hypoxic tumour zones. Indeed, increased NHE1 expression was observed in well perfused/peripheral regions of human breast cancers [40] and rat brain tumours [41]. Likewise, NHE1-dependent pHi regulation was reduced during in vitro hypoxia manipulations [42] and we have observed apparent reductions in hypoxic NHE1 activity in survival assays based on NH4Cl induced acidosis (unpublished results). Despite these suggestions for altered NHE1 activity in hypoxia, the significant impact of NHE1-ko on decreasing in vivo tumour growth described above [36, 39] support further attention on NHE1 manipulation for therapeutic applications.

(ii) Monocarboxylate Transporters (MCTs) MCTs are promiscuous transporters, which couple the movement of monocarboxylates (including lactate) with H+ and are integral to the maintenance of increased glycolytic activity [43]. However, as these proteins are driven primarily by the monocarboxylate substrate availability, they have not been defined as „classical‟ pHi regulating proteins. Consequently, until recently MCTs were normally overlooked

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in the context of overall tumour pHi regulation. As tumour cells produce excessive amounts of both lactate and H+ compared to normal cells, excretion of lactic acid via MCTs can provide important contributions to the resting tumour pHi (Figure 2). In the past decade, the impetus for studying MCTs in cancer related to the discovery of hypoxic-induction of MCT4 [44] and the need to understand lactate secretion mechanisms that permit continued glycolytic metabolism. This interest in MCTs also opened a new appreciation for pHi regulation. Fibroblast cells expressing MCT4 were used to confirm the contribution to resting pHi levels using both in vitro and in vivo measurements [45, 46]. Dramatic decreases in resting pHi levels due to disruption of activity were further confirmed via genetic knockout studies in cancer cell lines where it was revealed that a combined disruption of MCT1/2&4 was required [47]. Currently the MCT1/2 inhibitor (AZ3965) is progressing through clinical trials due to their promising anti-cancer properties in an extensive series of preclinical studies (for a recent extensive review see [48]). We predict that the outcome of these studies will be due to a combined result of MCT disruption on both cellular metabolism and maintenance of favourable pHi set points. In addition, the influence of lactic acid on components of the immune response has become an important area of research. Decreased cytotoxicity due to lactic acid in both T-cells [49] and NK-cells [50] has been reported along with modulation of dendritic cell function [51]. The role of lactic acid (linked to high LDHA expression) in supressing immune surveillance of melanomas by NK and T cells in a mouse model has been demonstrated recently [52]. The lactic acid concentration found in tumour pathophysiology dramatically reduced INF-y production and was proposed as a major means for tumours to escape the immune response [52]. Currently, as various immune-therapy strategies are providing the most promising curative potential in

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oncology [53], understanding the role of lactic acid and the means to manipulate it in the tumour environment will be essential for improved therapeutic efficacy (Figure 2). Thus, MCTs ability to modulate both concentrations of lactate and H+ (influencing both pHi and pHe) should hold promise as adjunct therapeutic strategies in addition to their direct effects on cellular metabolism.

CO2/HCO3- regulating proteins (i) Carbonic Anhydrases (CAs) CAs surged to the forefront in tumour pH regulatory studies following characterisation of the hypoxia-inducible (HIF-1), extracellular facing CA9 in tumours [54, 55]; a protein that is normally only expressed in limited regions of the gastro-intestinal tract. Confirmation of the role for CA9 in acidification of the extracellular tumour space due to the conversion of CO2 to H+ and HCO3- was followed by the demonstration that CA9 also assists in the maintenance of an alkaline pHi [56-59] (Figure 2). CA9 was further demonstrated to play an essential role in tumour cell proliferation in vitro and in vivo tumour xenograft growth [56, 60]. Of note, knockdown (kd) of CA9 tended to slow progression of tumour xenografts but tumours ultimately grew at reasonably rapid-rates albeit with a delay. These results have been recently extended using complete CA9 knockout cells [39]. Conversely, in syngeneic mouse breast cancer (4T1) models, CA9-kd alone showed impressive tumour regression [61]. Contributions from these pre-clinical studies and others inspired the race to develop pharmacological blocking agents for this essential protein. A recent large-scale meta-analysis (combining 147 independent studies) has confirmed that high CA9 expression acts as a tumour independent marker of poor prognosis [62]. Thus the rationale for identifying patients with high HIF-1 and CA9

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expression [55] and the subsequent application of CA9 inhibitors is strong. Substantial progress has been made for CA inhibitors with current clinical trials ongoing for CA9 inhibition [63]. Conclusions from these clinical trials will markedly shape future directions in this research area. In addition to CA9, CA12, which is an additional extracellular CA that exhibits hypoxia induction in certain cells [54], has recently received increased attention. CA12 has intriguingly been described as a good prognostic factor in lung and breast cancer [64, 65] despite possessing a similar physiological function to CA9. CA12 monoclonal antibodies have been shown to prevent cell proliferation (A549) in vitro and in xenografts [66]. Furthermore, increased CA12 expression and pHi alterations were found to contribute to chemotherapy resistance (doxorubicin) in multiple tumour cell lines [67]. Observations of compensatory protein expression patterns of CA12 in response to CA9 gene interference [56, 68] combined with reduced antibody efficacy in cells expressing both CA9 and CA12 [66] has led to the suggestion that inhibition of multiple CA isoforms may be more efficient than continued pursuit of single CA inhibition. Recently, this has been further observed in a complete genomic knockout model of CA9 where significant upregulation of CA12 (and also CA2) was implicated in the ability to promote tumour xenograft growth [39]. Further development of multiple CA isoform knockout models are being pursued and should aid greatly in the continued pharmacology development process. These knockout models should additionally uncover the importance of intra vs. extra cellular CAs in the directional movement of metabolically produced CO2 and shed light on differential prognostic factors proposed for different CA isoforms. Extensive analysis of CAs in normal cell physiology have indicated important contributions of both compartments to create a “push and pull” mechanism for CO2 transport [69-71] and therefore it will be essential

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to determine the contribution of this mechanism in tumour cells for improved understanding of CA drug inhibitors as an anti-cancer strategy. Research on CA9 has now branched into other areas of oncology including the development of cancer cell „stemness‟. Cancer stem cells (CSCs) and circulating tumour cells (CTCs) are recognised as key components of the metastatic phenotype and ultimately provide a major obstacle in the complete eradication of patient tumours (for recent review see [72]). CSCs inhabit specialised environmental „niches‟ containing various signalling components and cytokines required to maintain a balance between differentiation and proliferation [72]. The hypoxic niche found in tumours has been established to contribute to the CSC phenotype in a variety of cancer types [73-77]. Mechanistically, HIF plays an important role in the hypoxia CSC phenotype [77], and hypoxia related epigenetic suppression of DICER expression has also been reported to favour breast CSCs development [78]. Since CA9 is one of the most potent hypoxia targets in cancer, it became of interest to investigate in the context of CSC formation. Initially, CA9 was determined to not play a role in colorectal CSCs differentiation [76]. However, follow up studies on murine (4T1) and human (MDA-MB-231) breast cancer models strongly suggested that CA9 was required for expansion of the hypoxic CSC population [79]. Use of CA9 small molecule inhibitors further indicated that the catalytic activity of CA9 is required for maintenance of the CSC niche although pH alterations were not directly implicated [79]. This work was recently extended to show that CA9 contributes to the production of chemokines and cytokines that are important in the metastatic niche [80]. In addition, CA9 expression in CSCs was shown to predict sensitivity to chemotherapeutic agents such as HDAC inhibitors [81]. Acidosis alone was shown independently to promote the CSC phenotype in glioma cells [82]. This has been

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most recently confirmed by observations of a synergistic effect of hypoxia and acidosis on the maintenance of CSCs, with a mechanism involving HSP90 and HIFs [83]. Although the promise of inducing stem cell phenotypes with acidic stress reported in 2014 became mired in controversy due to non-reproducibility and paper retractions [84], acidosis appears to directly contribute to maintenance of the CSC niche (Figure 2). Thus, contributions of CA9 (and other CAs) to tumour acidosis are of great interest in the progressive understanding of CSC contributions to tumour progression. Finally, CAs have received attention due to their contributions to other membrane transport mechanisms. Intracellular CA2 facilitates MCT1/4 transport in a non-catalytic function via presenting what has been described as „proton antenna‟ [85]. Functionally, this is explained by a binding of CA2 to the C-terminal region of MCT4 [86] that enables rapid H+ substrate availability from protonated sites on CA2 (as compared to slow cytosolic H+-diffusion) for co-transport with lactate via MCT4. This concept of H+-diffusion limitations was further demonstrated by the molecular „proton antenna fusion‟ to MCT4 [87]. Furthermore, the presence of both intracellular (CA2) and extracellular (CA4) was shown to enhance MCT transport in an additive (but non-catalytic) fashion [88]. This additive effect was attributed to the dissipation of H+-microdomains on either side of the membrane to enhance overall transport efficiency. The overall CA enhanced MCT transport model follows the principle described above as a „push and pull‟ mechanism for CAs in moving CO 2 across cell membranes [69-71]. Thus, in addition to its catalytic activity related to CO2 conversion, tumour CA likely contributes significantly to the set point of tumour cell pH via enhancement of MCT activity.

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(iii) Bicarbonate Transporters Tumour cell bicarbonate uptake has been central to the proposed mechanism of extracellular CA control of pHi and pHe. HCO3- produced at the extracellular surface is proposed to be rapidly „re-captured‟ by the tumour cell in order to assist in cellular buffering to guard against extracellular acidosis (for extensive discussion see [3, 4, 89]) (Figure 1&2). Furthermore, an extensive in vitro pHi regulating characterisation of tumour cells demonstrated a shift away from predominantly H+transport processes towards HCO3- dependent transport mechanisms [42]. Identification of the specific bicarbonate transporters involved in tumour cells remained elusive until 2009 when the electroneutral Na+/HCO3- co-transporter (NBCn1/SLC4A7) was implicated in breast cancer progression [90]. This was followed by an extensive series of studies showing that indeed SLC4A7 plays an important role in pHi regulation and tumour cell progression in breast cancers [38, 40, 91-94]. A recent SLC4A7 knockout mouse model has reinforced its importance via demonstration of delayed breast cancer development following chemical induction (using 7,12-dimethylbenz(α)anthracene (DMBA)) and a reduction in tumour size [95]. Knockout of SLC4A7 caused pHi acidification demonstrating a key role for this transporter in the maintenance of a resting pHi that is amenable to cellular progression and reduction of cell proliferation in vitro was observed [95]. This recent study, presenting for the first time a causal link between SLC4A7 and breast cancer progression, will warrant attention in the context of future therapeutic development. Additionally, the molecular search for important tumour cell bicarbonate transporters was investigated by QPCR screening for potential hypoxia induction in all of the described bicarbonate transport family members [96]. The electrogenic NBC (SLC4A4) gene was strongly induced in hypoxia in colon adenocarcinoma cells

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providing excitement that this could provide a functional hypoxia-induced complex with CA9. However, hypoxia induction of SLC4A4 was not found to be a generalised phenotype in other cancer cell types [96]. Nonetheless, gene interference studies in colon and breast cancer cells indicated an important role for SLC4A4 in the areas of pHi regulation, cell proliferation and cell migration [96]. A recent study has extended the work on hypoxia induction of SLC4A4 and indicated a wider range of differential bicarbonate transporter induction patterns (with variations between cell lines) upon longer and more severe hypoxia exposure [97]. This study also revealed that SLC4A9, a transporter who‟s function has been controversial in past literature [98], was induced in colon, breast and brain cancer cells [97]. Knockdown of SLC4A9 dramatically reduced tumour xenograft formation [97]. Thus, considering the series of studies described above, it seems clear that tumour cells utilise a Na+/HCO3- co-transport (NBC) mechanism for HCO3- import and regulation of pHi. The molecular NBC identity may vary (SLC4 A4, A7, A9) between tumour types and either be constitutively expressed or regulated by hypoxia with a likelihood that multiple NBC isoforms are expressed in a redundant manner to ensure efficient pHi regulation. Pharmacological blockers of NBCs are notoriously nonspecific [99] and have been hampered by a lack of crystal structure data. However, improvements are being made and potentially a more broad-spectrum NBC inhibitor will prove to be the most useful for anti-cancer strategies targeting the pHi regulating component of bicarbonate transport. These inhibitors should effectively lower pHi and maintain HCO3- in the extracellular space to help buffer acidic pHe, both components of which would benefit cancer treatment strategies. Another aspect of bicarbonate transport that has been recently highlighted involves the differential SLC4A2 (Anion exchanger, AE2) expression found in

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fibroblasts (stroma) compared to tumour cells [100]. This has been shown to provide a means for absorption of tumour cell generated acidity that can then be effectively vented towards the vasculature via fibroblast gap-junction proteins (Cx43) and diffusible buffers [100]. The contribution of tumour cell generated acidity and fibroblast cooperation to in vivo tumour pH regulation will be of great interest in future studies. This acid-venting mechanism would be potentially disrupted by either the CA inhibition or buffer therapy strategies discussed above and below.

(iii) Buffer/HCO3- Therapy Development The group of Gatenby and Gillies has pursued the therapeutic potential of using a buffer therapy strategy to counteract tumour acidity (Figure 2). They first demonstrated that utilisation of „buffer therapy‟ in the form of bicarbonate acts to increase tumour pHe without an appreciable effect on systemic pH via a „compensated metabolic alkalosis‟ [101]. Past concerns have been raised over the chronic use of bicarbonate due to detrimental effects arising from disrupted pH homeostasis however it is important to note that these observations typically stemmed from an uncontrolled „self-dosing‟ of bicarbonate [102]. Therefore, demonstration of controlled bicarbonate therapy specifically targeting tumour pHe without harming systemic pH is reassuring. Outstanding effects on the prevention of metastases in pre-clinical models were initially observed when tumour acidity was neutralised via administration of buffer therapy (either NaHCO3 or synthetic buffers) [103, 104]. Focus in the use of buffer therapy has now shifted to interactions with the tumour immune response. As discussed above in the MCT section, extracellular acidosis is associated with a blunting of the immune response [105]. Recent work has shown the importance of buffer therapy in cancer models that are associated

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with increased levels of infiltrating T-cells [106]. In addition, substantial improvements for 3 promising immune-therapy strategies (anti-CTLA-4, anti-PD1, or adoptive T-cell transfer) were observed with bicarbonate therapy [106]. Neutralisation of tumour acidity via „buffer therapy‟ or CA/NBC inhibition may thus prove useful as a future adjuvant treatment for promising curative immune-therapies. Bicarbonate therapy is also being considered for use in targeting mTORC1, the master regulator of cell proliferation. mTORC1 activity decreases during acidosis and it was recently demonstrated that cells lose sensitivity to rapamycin‟s (mTORC1 inhibitor) prevention of proliferation [107]. When experiments were scaled up to in vivo tumour treatment (xenografts/allografts), bicarbonate therapy substantially improved the efficacy of rapamycin treatment [107]. This same research group had also recently demonstrated that both disruption of CA9 (using shRNA) or application of the broad CA inhibitor acetazolamide improves the anti-proliferative effect of rapamycin [108]. However, these data were discussed in the context of hypoxia limitations on mTORC1, thus CA9 disruption would target hypoxic areas to synergise with the effects of rapamycin in normoxic areas [108]. Considering that CA9 disruption and acetazolamide could lead to elevation of tumour pH e it is possible that a similar effect was being observed on mTORC1 inhibition between CA9 disruption studies and bicarbonate therapy approaches. As mTORC1 inhibition is of great interest for clinical applications, further understanding of interactions with tumour acidity may assist in improving the efficacy of mTORC1 inhibitors. Other areas of tumour acidity that would be sensitive to buffer therapy and remain to be extensively investigated include acid-sensing mechanisms (for review see [109]). The acid-sensing ion channel 1 (ASIC1) has just been implicated in breast cancer growth and metastasis [110]. However, as ASIC1 was localised to the

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mitochondria and cytoplasm [110], cellular compartments that are shown to be alkaline in tumour cells, the role of ASIC1 (and other pH sensors) in sensing external tumour acidity remains to be clarified.

Summary and Perspectives Although initial projections to attack tumour cells via simple pHi disruptions have proven elusive, often due to redundancy in the expression of pHi regulating proteins, reasons for optimism exist with recent advances in the field. Now that reductions in tumour growth have been established upon disruption of numerous pH regulating proteins including NHEs, MCTs, NBCs and CAs, projections as to how these proteins can be properly exploited in combination or as adjunct strategies with classical and developing oncology therapies will be the focus of future work. The current rapid expansion of genomically altered cell and animal models will greatly assist pharmacological progression in this area. Furthermore, improved knowledge of the overall role of acidosis in the tumour environment has assisted in the development of the non-protein targeted „buffer-therapy‟ strategy that may prove to be an effective means to improve patient response to game-changing immunotherapies. Recent progress in the ability to monitor multiple cell interactions in the context of tumour acidity will evidently push the field forward to be better positioned in the context of clinical applications. Much work remains, however it appears that the defining acidic nature of tumours and their unique protein complement that enables adaptation within their environment present continued reasons for excitement in the development of improved anti-cancer strategies.

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Acknowledgements SKP is funded by the Centre Scientifique de Monaco (CSM). In addition to the CSM, JP received funding from GEMLUC and is supported by the Institute for Research on Cancer and Aging (IRCAN), CNRS, INSERM, Centre A. Lacassagne, University of Nice-Sophia Antipolis and the Ligue Nationale Contre le Cancer (LNCC).

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Figure Legends

Figure 1. Generalised model of tumour generated acidity and pH regulation. Oncogenic transformation and subsequent gene regulation via hypoxia drive tumour cells to produce increased levels of metabolic acid in the form of H+ and CO2. Intracellular pH (pHi) is in turn regulated by contributions from cellular buffers and membrane transporting mechanisms that ultimately excrete acid and import base resulting in stable pHi and acidic extracellular pH (pHe). For illustrative purposes, red and blue colour coding is used to represent acidic (red) and basic (blue) contributing components of tumour cell pH regulation.

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Figure 2. Tumour cell pH regulating proteins under investigation for therapeutic development. A combination of hypoxia/HIF-regulated (red) and normoxia-regulated (green) proteins are utilised to achieve efficient regulation of pHi and acidification of pHe. These include varying isoforms from the Na+/H+ exchangers (NHEs), monocarboxylate transporters (MCTs), carbonic anhydrases (CA9/12) and Na+/HCO3- co-transporters (NBCs) families. Targeting of these individual proteins reduce tumour cell progression. Acidification of pHe promotes favourable „nichepriming‟ for cancer stem cells (CSCs) and inhibition of infiltrating immune cells. Application of buffer therapy and inhibition of pH regulating membrane proteins to increase pHe provide potential means to limit CSCs and enhance immune-targeted and mTORC1 inhibiting therapies. Membrane-bound and cytoplasmic pH sensors remain to be determined for their therapeutic relevance in targeting the control mechanisms of tumour cell acidity. 19

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