Amino Acid Transporters Are a Vital Focal Point in the Control of mTORC1 Signaling and Cancer.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates signals from growth factors and nutrients to control biosynthetic processes, including protein, lipid, and nucleic acid synthesis. Dysregulation in the mTORC1 network underlies a wide array of pathological states, including metabolic diseases, neurological disorders, and cancer. Tumor cells are characterized by uncontrolled growth and proliferation due to a reduced dependency on exogenous growth factors. The genetic events underlying this property, such as mutations in the PI3K-Akt and Ras-Erk signaling networks, lead to constitutive activation of mTORC1 in nearly all human cancer lineages. Aberrant activation of mTORC1 has been shown to play a key role for both anabolic tumor growth and resistance to targeted therapeutics. While displaying a growth factor-independent mTORC1 activity and proliferation, tumors cells remain dependent on exogenous nutrients such as amino acids (AAs). AAs are an essential class of nutrients that are obligatory for the survival of any cell. Known as the building blocks of proteins, AAs also act as essential metabolites for numerous biosynthetic processes such as fatty acids, membrane lipids and nucleotides synthesis, as well as for maintaining redox homeostasis. In most tumor types, mTORC1 activity is particularly sensitive to intracellular AA levels. This dependency, therefore, creates a targetable vulnerability point as cancer cells become dependent on AA transporters to sustain their homeostasis. The following review will discuss the role of AA transporters for mTORC1 signaling in cancer cells and their potential as therapeutic drug targets.

Genetic Ablation of the Cystine Transporter xCT in PDAC Cells Inhibits mTORC1, Growth, Survival, and Tumor Formation via Nutrient and Oxidative Stresses.

Although chemoresistance remains a primary challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC), exploiting oxidative stress might offer novel therapeutic clues. Here we explored the potential of targeting cystine/glutamate exchanger (SLC7A11/xCT), which contributes to the maintenance of intracellular glutathione (GSH). Genomic disruption of xCT via CRISPR-Cas9 was achieved in two PDAC cell lines, MiaPaCa-2 and Capan-2, and xCT-KO clones were cultivated in the presence of N-acetylcysteine. Although several cystine/cysteine transporters have been identified, our findings demonstrate that, in vitro, xCT plays the major role in intracellular cysteine balance and GSH biosynthesis. As a consequence, both xCT-KO cell lines exhibited amino acid stress with activation of GCN2 and subsequent induction of ATF4, inhibition of mTORC1, proliferation arrest, and cell death. Tumor xenograft growth was delayed but not suppressed in xCT-KO cells, which indicated both the key role of xCT and also the presence of additional mechanisms for cysteine homeostasis in vivo. Moreover, rapid depletion of intracellular GSH in xCT-KO cells led to accumulation of lipid peroxides and cell swelling. These two hallmarks of ferroptotic cell death were prevented by vitamin E or iron chelation. Finally, in vitro pharmacologic inhibition of xCT by low concentrations of erastin phenocopied xCT-KO and potentiated the cytotoxic effects of both gemcitabine and cisplatin in PDAC cell lines. In conclusion, our findings strongly support that inhibition of xCT, by its dual induction of nutritional and oxidative cellular stresses, has great potential as an anticancer strategy. SIGNIFICANCE: The cystine/glutamate exchanger xCT is essential for amino acid and redox homeostasis and its inhibition has potential for anticancer therapy by inducing ferroptosis.

AKT1 restricts the invasive capacity of head and neck carcinoma cells harboring a constitutively active PI3 kinase activity.

BACKGROUND: In mammals, the AKT/PKB protein kinase family comprises three members (AKT1-3). PI3-Kinase (PI3K), a key oncogene involved in a wide variety of cancers, drives AKT activity. Constitutive activation of the PI3K/AKT pathway has been associated with tumorigenic properties including uncontrolled cell proliferation and survival, angiogenesis, promotion of cellular motility, invasiveness and metastasis. However, AKT1 activity has also been recently shown to repress the invasive properties of breast cancer cells in specific contexts. METHODS: This study used both pharmacological and shRNA approaches to inhibit AKT function, microscopy to characterize the cellular morphology, 3D spheroid models to assess migratory and invasive cellular capacities and a phenotypic screening approach based on electrical properties of the cells. RESULTS: Here we demonstrate that the alternative action of AKT1 on invasive properties of breast cancers can be extended to head and neck carcinomas, which exhibit constitutive activation of the PI3K/AKT pathway. Indeed, inhibition of AKT1 function by shRNA or a specific pharmacological inhibitor resulted in cellular spreading and an invasive phenotype. A phenotypic screening approach based on cellular electrical properties corroborated microscopic observations and provides a foundation for future high-throughput screening studies. This technique further showed that the inhibition of AKT1 signaling is phenocopied by blocking the mTORC1 pathway with rapamycin. CONCLUSION: Our study suggests that the repressive action of PI3K/AKT1 on cellular invasive properties may be a mechanism common to several cancers. Current and future studies involving AKT inhibitors must therefore consider this property to prevent metastases and consequently to improve survival.

The glutamine transporter ASCT2 (SLC1A5) promotes tumor growth independently of the amino acid transporter LAT1 (SLC7A5).

The transporters for glutamine and essential amino acids, ASCT2 (solute carrier family 1 member 5, SLC1A5) and LAT1 (solute carrier family 7 member 5, SLC7A5), respectively, are overexpressed in aggressive cancers and have been identified as cancer-promoting targets. Moreover, previous work has suggested that glutamine influx via ASCT2 triggers essential amino acids entry via the LAT1 exchanger, thus activating mechanistic target of rapamycin complex 1 (mTORC1) and stimulating growth. Here, to further investigate whether these two transporters are functionally coupled, we compared the respective knockout (KO) of either LAT1 or ASCT2 in colon (LS174T) and lung (A549) adenocarcinoma cell lines. Although ASCT2KO significantly reduced glutamine import (>60% reduction), no impact on leucine uptake was observed in both cell lines. Although an in vitro growth-reduction phenotype was observed in A549-ASCT2KO cells only, we found that genetic disruption of ASCT2 strongly decreased tumor growth in both cell lines. However, in sharp contrast to LAT1KO cells, ASCT2KO cells displayed no amino acid (AA) stress response (GCN2/EIF2a/ATF4) or altered mTORC1 activity (S6K1/S6). We therefore conclude that ASCT2KO reduces tumor growth by limiting AA import, but that this effect is independent of LAT1 activity. These data were further supported by in vitro cell proliferation experiments performed in the absence of glutamine. Together these results confirm and extend ASCT2's pro-tumoral role and indicate that the proposed functional coupling model of ASCT2 and LAT1 is not universal across different cancer types.

Disrupting the 'Warburg effect' re-routes cancer cells to OXPHOS offering a vulnerability point via 'ferroptosis'-induced cell death.

The evolution of life from extreme hypoxic environments to an oxygen-rich atmosphere has progressively selected for successful metabolic, enzymatic and bioenergetic networks through which a myriad of organisms survive the most extreme environmental conditions. From the two lethal environments anoxia/high O2, cells have developed survival strategies through expression of the transcriptional factors ATF4, HIF1 and NRF2. Cancer cells largely exploit these factors to thrive and resist therapies. In this review, we report and discuss the potential therapeutic benefit of disrupting the major Myc/Hypoxia-induced metabolic pathway, also known as fermentative glycolysis or "Warburg effect", in aggressive cancer cell lines. With three examples of genetic disruption of this pathway: glucose-6-phosphate isomerase (GPI), lactate dehydrogenases (LDHA and B) and lactic acid transporters (MCT1, MCT4), we illuminate how cancer cells exploit metabolic plasticity to survive the metabolic and energetic blockade or arrest their growth. In this context of NRF2 contribution to OXPHOS re-activation we will show and discuss how, by disruption of the cystine transporter xCT (SLC7A11), we can exploit the acute lethal phospholipid peroxidation pathway to induce cancer cell death by 'ferroptosis'.

Targeting pH regulating proteins for cancer therapy-Progress and limitations.

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.

Hypoxia and cellular metabolism in tumour pathophysiology.

Cancer cells are optimised for growth and survival via an ability to outcompete normal cells in their microenvironment. Many of these advantageous cellular adaptations are promoted by the pathophysiological hypoxia that arises in solid tumours due to incomplete vascularisation. Tumour cells are thus faced with the challenge of an increased need for nutrients to support the drive for proliferation in the face of a diminished extracellular supply. Among the many modifications occurring in tumour cells, hypoxia inducible factors (HIFs) act as essential drivers of key pro-survival pathways via the promotion of numerous membrane and cytosolic proteins. Here we focus our attention on two areas: the role of amino acid uptake and the handling of metabolic acid (CO2 /H+ ) production. We provide evidence for a number of hypoxia-induced proteins that promote cellular anabolism and regulation of metabolic acid-base levels in tumour cells including amino-acid transporters (LAT1), monocarboxylate transporters, and acid-base regulating carbonic anhydrases (CAs) and bicarbonate transporters (NBCs). Emphasis is placed on current work manipulating multiple CA isoforms and NBCs, which is at an interesting crossroads of gas physiology as they are regulated by hypoxia to contribute to the cellular handling of CO2 and pHi regulation. Our research combined with others indicates that targeting of HIF-regulated membrane proteins in tumour cells will provide promising future anti-cancer therapeutic approaches and we suggest strategies that could be potentially used to enhance these tactics.

Genetic disruption of the pHi-regulating proteins Na+/H+ exchanger 1 (SLC9A1) and carbonic anhydrase 9 severely reduces growth of colon cancer cells.

Hypoxia and extracellular acidosis are pathophysiological hallmarks of aggressive solid tumors. Regulation of intracellular pH (pHi) is essential for the maintenance of tumor cell metabolism and proliferation in this microenvironment and key proteins involved in pHi regulation are of interest for therapeutic development. Carbonic anhydrase 9 (CA9) is one of the most robustly regulated proteins by the hypoxia inducible factor (HIF) and contributes to pHi regulation. Here, we have investigated for the first time, the role of CA9 via complete genomic knockout (ko) and compared its impact on tumor cell physiology with the essential pHi regulator Na+/H+ exchanger 1 (NHE1). Initially, we established NHE1-ko LS174 cells with inducible CA9 knockdown. While increased sensitivity to acidosis for cell survival in 2-dimensions was not observed, clonogenic proliferation and 3-dimensional spheroid growth in particular were greatly reduced. To avoid potential confounding variables with use of tetracycline-inducible CA9 knockdown, we established CA9-ko and NHE1/CA9-dko cells. NHE1-ko abolished recovery from NH4Cl pre-pulse cellular acid loading while both NHE1 and CA9 knockout reduced resting pHi. NHE1-ko significantly reduced tumor cell proliferation both in normoxia and hypoxia while CA9-ko dramatically reduced growth in hypoxic conditions. Tumor xenografts revealed substantial reductions in tumor growth for both NHE1-ko and CA9-ko. A notable induction of CA12 occurred in NHE1/CA9-dko tumors indicating a potential means to compensate for loss of pH regulating proteins to maintain growth. Overall, these genomic knockout results strengthen the pursuit of targeting tumor cell pH regulation as an effective anti-cancer strategy.

The Central Role of Amino Acids in Cancer Redox Homeostasis: Vulnerability Points of the Cancer Redox Code.

A fine balance in reactive oxygen species (ROS) production and removal is of utmost importance for homeostasis of all cells and especially in highly proliferating cells that encounter increased ROS production due to enhanced metabolism. Consequently, increased production of these highly reactive molecules requires coupling with increased antioxidant defense production within cells. This coupling is observed in cancer cells that allocate significant energy reserves to maintain their intracellular redox balance. Glutathione (GSH), as a first line of defense, represents the most important, non-enzymatic antioxidant component together with the NADPH/NADP+ couple, which ensures the maintenance of the pool of reduced GSH. In this review, the central role of amino acids (AAs) in the maintenance of redox homeostasis in cancer, through GSH synthesis (cysteine, glutamate, and glycine), and nicotinamide adenine dinucleotide (phosphate) production (serine, and glutamine/glutamate) are illustrated. Special emphasis is placed on the importance of AA transporters known to be upregulated in cancers (such as system xc-light chain and alanine-serine-cysteine transporter 2) in the maintenance of AA homeostasis, and thus indirectly, the redox homeostasis of cancer cells. The role of the ROS varies (often described as a "two-edged sword") during the processes of carcinogenesis, metastasis, and cancer treatment. Therefore, the context-dependent role of specific AAs in the initiation, progression, and dissemination of cancer, as well as in the redox-dependent sensitivity/resistance of the neoplastic cells to chemotherapy are highlighted.

Metabolic Plasiticy in Cancers-Distinct Role of Glycolytic Enzymes GPI, LDHs or Membrane Transporters MCTs.

Research on cancer metabolism has recently re-surfaced as a major focal point in cancer field with a reprogrammed metabolism no longer being considered as a mere consequence of oncogenic transformation, but as a hallmark of cancer. Reprogramming metabolic pathways and nutrient sensing is an elaborate way by which cancer cells respond to high bioenergetic and anabolic demands during tumorigenesis. Thus, inhibiting specific metabolic pathways at defined steps should provide potent ways of arresting tumor growth. However, both animal models and clinical observations have revealed that this approach is seriously limited by an extraordinary cellular metabolic plasticity. The classical example of cancer metabolic reprogramming is the preference for aerobic glycolysis, or Warburg effect, where cancers increase their glycolytic flux and produce lactate regardless of the presence of the oxygen. This allows cancer cells to meet the metabolic requirements for high rates of proliferation. Here, we discuss the benefits and limitations of disrupting fermentative glycolysis for impeding tumor growth at three levels of the pathway: (i) an upstream block at the level of the glucose-6-phosphate isomerase (GPI), (ii) a downstream block at the level of lactate dehydrogenases (LDH, isoforms A and B), and (iii) the endpoint block preventing lactic acid export (MCT1/4). Using these examples of genetic disruption targeting glycolysis studied in our lab, we will discuss the responses of different cancer cell lines in terms of metabolic rewiring, growth arrest, and tumor escape and compare it with the broader literature.

Genetic Disruption of the Multifunctional CD98/LAT1 Complex Demonstrates the Key Role of Essential Amino Acid Transport in the Control of mTORC1 and Tumor Growth.

The CD98/LAT1 complex is overexpressed in aggressive human cancers and is thereby described as a potential therapeutic target. This complex promotes tumorigenesis with CD98 (4F2hc) engaging ß-integrin signaling while LAT1 (SLC7A5) imports essential amino acids (EAA) and promotes mTORC1 activity. However, it is unclear as to which member of the heterodimer carries the most prevalent protumoral action. To answer this question, we explored the tumoral potential of each member by gene disruption of CD98, LAT1, or both and by inhibition of LAT1 with the selective inhibitor (JPH203) in six human cancer cell lines from colon, lung, and kidney. Each knockout respectively ablated 90% (CD98 KO: ) and 100% (LAT1 KO: ) of Na(+)-independent leucine transport activity. LAT1 KO: or JPH203-treated cells presented an amino acid stress response with ATF4, GCN2 activation, mTORC1 inhibition, and severe in vitro and in vivo tumor growth arrest. We show that this severe growth phenotype is independent of the level of expression of CD98 in the six tumor cell lines. Surprisingly, CD98 KO: cells with only 10% EAA transport activity displayed a normal growth phenotype, with mTORC1 activity and tumor growth rate undistinguishable from wild-type cells. However, CD98 KO: cells became extremely sensitive to inhibition or genetic disruption of LAT1 (CD98 KO: /LAT1 KO: ). This finding demonstrates that the tumoral potential of CD98 KO: cells is due to residual LAT1 transport activity. Therefore, these findings clearly establish that LAT1 transport activity is the key growth-limiting step of the heterodimer and advocate the pharmacology development of LAT1 transporter inhibitors as a very promising anticancer target. Cancer Res; 76(15); 4481-92. ©2016 AACR.

Hypoxia optimises tumour growth by controlling nutrient import and acidic metabolite export.

In their quest for survival and successful growth, cancer cells optimise their cellular processes to enable them to outcompete normal cells in their microenvironment. In essence cancer cells: (i) enhance uptake of nutrients/metabolites, (ii) utilise nutrients more efficiently via metabolic alterations and (iii) deal with the metabolic waste products in a way that furthers their progression while hampering the survival of normal tissue. Hypoxia Inducible Factors (HIFs) act as essential drivers of these adaptations via the promotion of numerous membrane proteins including glucose transporters (GLUTs), monocarboxylate transporters (MCTs), amino-acid transporters (LAT1, xCT), and acid-base regulating carbonic anhydrases (CAs). In addition to a competitive growth advantage for tumour cells, these HIF-regulated proteins are implicated in metastasis, cancer 'stemness' and the immune response. Current research indicates that combined targeting of these HIF-regulated membrane proteins in tumour cells will provide promising therapeutic strategies in the future.

The Na(+)/HCO3(-) Co-Transporter SLC4A4 Plays a Role in Growth and Migration of Colon and Breast Cancer Cells.

The hypoxic and acidic tumor environment necessitates intracellular pH (pHi) regulation for tumor progression. Carbonic anhydrase IX (CA IX; hypoxia-induced) is known to facilitate CO2 export and generate HCO3(-) in the extracellular tumor space. It has been proposed that HCO3(-) is re-captured by the cell to maintain an alkaline pHi . A diverse range of HCO3(-) transporters, coupled with a lack of a clear over-expression in cancers have limited molecular identification of this cellular process. Here, we report that hypoxia induces the Na(+)/HCO3(-) co-transporter (NBCe1) SLC4A4 mRNA expression exclusively in the LS174 colon adenocarcinoma cell line in a HIF1α dependent manner. HCO3(-) dependent pHi recovery observations revealed the predominant use of an NBC mechanism suggesting that reversal of a Cl(-)/HCO3(-) exchanger is not utilized for tumor cell pHi regulation. Knockdown of SLC4A4 via shRNA reduced cell proliferation and increased mortality during external acidosis and spheroid growth. pHi recovery from acidosis was partially reduced with knockdown of SLC4A4. In MDA-MB-231 breast cancer cells expressing high levels of SLC4A4 compared to LS174 cells, SLC4A4 knockdown had a strong impact on cell proliferation, migration, and invasion. SLC4A4 knockdown also altered expression of other proteins including CA IX. Furthermore the Na(+)/HCO3(-) dependent pHi recovery from acidosis was reduced with SLC4A4 knockdown in MDA-MB-231 cells. Combined our results indicate that SLC4A4 contributes to the HCO3(-) transport and tumor cell phenotype. This study complements the on-going molecular characterization of the HCO3(-) re-uptake mechanism in other tumor cells for future strategies targeting these potentially important drug targets.

Disrupting proton dynamics and energy metabolism for cancer therapy.

Intense interest in the 'Warburg effect' has been revived by the discovery that hypoxia-inducible factor 1 (HIF1) reprogrammes pyruvate oxidation to lactic acid conversion; lactic acid is the end product of fermentative glycolysis. The most aggressive and invasive cancers, which are often hypoxic, rely on exacerbated glycolysis to meet the increased demand for ATP and biosynthetic precursors and also rely on robust pH-regulating systems to combat the excessive generation of lactic and carbonic acids. In this Review, we present the key pH-regulating systems and synthesize recent advances in strategies that combine the disruption of pH control with bioenergetic mechanisms. We discuss the possibility of exploiting, in rapidly growing tumours, acute cell death by 'metabolic catastrophe'.

Hypoxia promotes tumor cell survival in acidic conditions by preserving ATP levels.

The efficacy of targeting pH disruption to induce cell death in the acidic and hypoxic tumor microenvironment continues to be assessed. Here we analyzed the impact of varying levels of hypoxia in acidic conditions on fibroblast and tumor cell survival. Across all cell lines tested, hypoxia (1% O(2)) provided protection against acidosis induced cell death compared to normoxia. Meanwhile severe hypoxia (0.1% O(2)) removed this protection and in some cases exacerbated acidosis-induced cell death. Differential survival between cell types during external acidosis correlated with their respective intracellular pH regulating capabilities. Cellular ATP measurements were conducted to determine their contribution to cell survival under these combined stresses. In general, hypoxia (1% O(2)) maintained elevated ATP levels in acidic conditions while severe hypoxia did not. To further explore this interaction we combined acidosis with ATP depletion using 2-deoxyglucose and observed an enhanced rate of cell mortality. Striking results were also observed with hypoxia providing protection against cell death in spite of a severe metabolic stress induced by a combination of acidosis and oligomycin. Finally, we demonstrated that both HIF1α and HIF2α expression were drastically reduced in hypoxic and acidic conditions indicating a sensitivity of this protein to cellular pH conditions. This knockdown of HIF expression by acidosis has implications for the development of therapies targeting the disruption of cellular pH regulation. Our results reinforce the proof of concept that acidosis and metabolic disruption affecting ATP levels could be exploited as a tumor cell killing strategy.

Knock-down of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis.

The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases (CA) IX and CAXII constitute a robust intracellular pH (pH(i))-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX) and LS174Tr cells (inducible knock-down for ca9/ca12) were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pH(o) manipulations and hypoxia (1% O(2)) exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pH(i)-regulating capacity of fibroblasts through inhibition of Na(+)/H(+) exchanger 1 sensitize cells to radiation-induced cell death. Secondly, the pH(i)-regulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50 and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pH(i) regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pH(i)-regulating CAs as an anti-cancer strategy.

Circulating tumor cells in prostate cancer: a potential surrogate marker of survival.

Prostate-specific antigen (PSA) levels in blood are widely used in prostate cancer (PCa) for the management of this disease at every stage of progression. Currently, PSA levels combined with clinical stage and Gleason score provide the best predictor of survival and the main element to monitor treatment efficiency. However, these areas could be improved by utilizing emerging biomarkers. Recently, circulating tumor cells (CTCs) and disseminating tumor cells (DTCs) have been detected in PCa and may be a new surrogate candidate. Here we provide a systematic review of the literature in order to describe the current evidence of CTC/DTC surrogacy regarding outcome of prostate cancer patients. We also discuss several markers that could be used to increase the sensitivity and specificity of CTC/DTC detection. CTC/DTC detection is performed using a wide variety of techniques. Initially, reverse transcriptase polymerase chain reaction (RT-PCR) based methods were utilized with weak correlation between their positive detection and patients' outcome. More recent immunological techniques have indicated a reproducible correlation with outcome. Such surrogate markers may enable clinicians to provide early detection for inefficient treatments and patients with poor prognosis that are candidates for treatment intensification. Dissecting the micrometastasis phenomenon in CTCs/DTCs is a key point to increase surrogacy of this biomarker.

pH control mechanisms of tumor survival and growth.

A distinguishing phenotype of solid tumors is the presence of an alkaline cellular feature despite the surrounding acidic microenvironment. This phenotypic characteristic of tumors, originally described by Otto Warburg, arises due to alterations in metabolism of solid tumors. Hypoxic regions of solid tumors develop due to poor vascularization and in turn regulate the expression of numerous genes via the transcription factor HIF-1. Ultimately, the tumor microenvironment directs the development of tumor cells adapted to survive in an acidic surrounding where normal cells perish. The provision of unique pH characteristics in tumor cells provides a defining trait that has led to the pursuit of treatments that target metabolism, hypoxia, and pH-related mechanisms to selectively kill cancer cells. Numerous studies over the past decade involving the cancer-specific carbonic anhydrase IX have re-kindled an interest in pH disruption-based therapies. Although an acidification of the intracellular compartment is established as a means to induce normal cell death, the defining role of acid-base disturbances in tumor physiology and survival remains unclear. The aim of this review is to summarize recent data relating to the specific role of pH regulation in tumor cell survival. We focus on membrane transport and enzyme studies in an attempt to elucidate their respective functions regarding tumor cell pH regulation. These data are discussed in the context of future directions for the field of tumor cell acid-base-related research.

PKC mediates GnRH activation of a Na+/H+ exchanger in goldfish somatotropes.

Previous results suggest that gonadotropin-releasing hormone (GnRH) stimulation of somatotropin secretion in goldfish involves activation of Na(+)/H(+) exchange (NHE). We tested the hypothesis that GnRH alkalinizes intracellular pH (pH(i)) via protein kinase C (PKC) activation of NHE. Two types of alkalinization responses were observed in identified goldfish somatotropes preloaded with the pH-sensitive dye BCECF; the rate of pH(i) changes went from a neutral or slightly negative slope to either a positive or a less negative slope relative to control. Two GnRHs, the PKC-activating TPA, and dioctanoyl glycerol each caused an alkalinization in 70-90% of somatotropes. The PKC inhibitors, Bis II and Gö6976, the NHE inhibitor amiloride, or Na(+)-free solution attenuated TPA and GnRHs actions, suggesting that PKC mediates GnRH activation of NHE. Since amiloride and Na(+)-free solution caused acidification in somatotropes at rest, regulation of basal pH(i) in these cells likely involves Na(+) flux through amiloride-sensitive NHE.

Intracellular pH regulation in isolated trout gill mitochondrion-rich (MR) cell subtypes: evidence for Na+/H+ activity.

We have studied intracellular pH (pH(i)) recovery in isolated trout gill mitochondrion-rich (MR) cells following acidification by the NH(4)Cl pre-pulse technique. Within a mixed MR cell population, one cell type displayed Na(+)-independent pH(i) recovery while the other cell type lacked a Na(+)-independent pH(i) recovery. Cells displaying Na(+) independent recovery exhibited a significantly higher buffering capacity compared to cells lacking Na(+)-independent pH(i) recovery. Cells displaying Na(+) independent recovery were identified as PNA(+) (peanut lectin agluttinin binding) MR cells while those unable to recover were identified as PNA(-) (non-peanut lectin agluttinin binding) MR cells. Therefore, recovery from acidification in the absence of Na(+) provides a direct functional marker for PNA(+) and PNA(-) MR cells. Re-addition of Na(+) to acidified cells resulted in a transient pH(i) recovery in both cell types. This event was abolished by amiloride (500 microM) but it was insensitive to phenamil (50 microM). The phorbol ester PMA (1 microM) potentiated the Na(+) induced pH(i) recovery suggesting that activation by PKC is required for continuous Na(+)/H(+) exchanger activity in trout gill MR cells. This study is the first functional description of pH(i) recovery in lectin-identified trout gill MR cells and provides insight into a putative cellular signaling mechanism that may control pH(i) regulation in the gill epithelium.