Cancer cell behaviors mediated by dysregulated pH dynamics at a glance.

Dysregulated pH is a common characteristic of cancer cells, as they have an increased intracellular pH (pHi) and a decreased extracellular pH (pHe) compared with normal cells. Recent work has expanded our knowledge of how dysregulated pH dynamics influences cancer cell behaviors, including proliferation, metastasis, metabolic adaptation and tumorigenesis. Emerging data suggest that the dysregulated pH of cancers enables these specific cell behaviors by altering the structure and function of selective pH-sensitive proteins, termed pH sensors. Recent findings also show that, by blocking pHi increases, cancer cell behaviors can be attenuated. This suggests ion transporter inhibition as an effective therapeutic approach, either singly or in combination with targeted therapies. In this Cell Science at a Glance article and accompanying poster, we highlight the interconnected roles of dysregulated pH dynamics in cancer initiation, progression and adaptation.

A Histidine Cluster in the Cytoplasmic Domain of the Na-H Exchanger NHE1 Confers pH-sensitive Phospholipid Binding and Regulates Transporter Activity.

The Na-H exchanger NHE1 contributes to intracellular pH (pHi) homeostasis in normal cells and the constitutively increased pHi in cancer. NHE1 activity is allosterically regulated by intracellular protons, with greater activity at lower pHi However, the molecular mechanism for pH-dependent NHE1 activity remains incompletely resolved. We report that an evolutionarily conserved cluster of histidine residues located in the C-terminal cytoplasmic domain between two phosphatidylinositol 4,5-bisphosphate binding sites (PI(4,5)P2) of NHE1 confers pH-dependent PI(4,5)P2 binding and regulates NHE1 activity. A GST fusion of the wild type C-terminal cytoplasmic domain of NHE1 showed increased maximum PI(4,5)P2 binding at pH 7.0 compared with pH 7.5. However, pH-sensitive binding is abolished by substitutions of the His-rich cluster to arginine (RXXR3) or alanine (AXXA3), mimicking protonated and neutral histidine residues, respectively, and the RXXR3 mutant had significantly greater PI(4,5)P2 binding than AXXA3. When expressed in cells, NHE1 activity and pHi were significantly increased with NHE1-RXXR3 and decreased with NHE1-AXXA3 compared with wild type NHE1. Additionally, fibroblasts expressing NHE1-RXXR3 had significantly more contractile actin filaments and focal adhesions compared with fibroblasts expressing wild type NHE1, consistent with increased pHi enabling cytoskeletal remodeling. These data identify a molecular mechanism for pH-sensitive PI(4,5)P2 binding regulating NHE1 activity and suggest that the evolutionarily conserved cluster of four histidines in the proximal cytoplasmic domain of NHE1 may constitute a proton modifier site. Moreover, a constitutively activated NHE1-RXXR3 mutant is a new tool that will be useful for studying how increased pHi contributes to cell behaviors, most notably the biology of cancer cells.

Dynamic cell shapes and contacts in the developing Drosophila retina are regulated by the Ig cell adhesion protein hibris.

Cell shapes and contacts are dynamically regulated during organogenesis to enable contacts with relevant neighboring cells at appropriate times. During Drosophila larval eye development, an apical contact is established between one pair of non-neuronal cones cells, precluding contact between the opposing pair. Concurrent with changes in cell shape, these contacts reverse in early pupal life. The reversal in cone cell contacts occurs in a posterior to anterior gradient across the eye, following the developmental gradient established in the larval eye imaginal disc. Hibris (Hbs), an Immunoglobulin cell adhesion molecule homologous to vertebrate Nephrin, is required for cone cell morphogenesis. In hbs null mutants, a majority of cone cells fail to both establish wild-type contacts and achieve mature cone cell shapes. hbs acts cell autonomously in the cone cells to drive these changes. The work presented here indicates hbs contributes to the remodeling of cell contacts and cell shapes throughout development.

Oncogenic ß-catenin mutations evade pH-regulated degradation.

ß-catenin has roles in cell-cell adhesion and Wnt signaling. We recently showed that ß-catenin protein abundance is decreased at higher intracellular pH (pHi), mediated by pH-sensitive interaction with the beta-transducin repeat containing E3 ubiquitin protein ligase (ß-TrCP). Increased pHi facilitates ß-TrCP binding and degradation of ß-catenin. ß-catenin mutations that abrogate the pH-sensitive interaction induce significant tumors not seen with other ß-catenin stabilizing mutants.

ß-Catenin is a pH sensor with decreased stability at higher intracellular pH.

ß-Catenin functions as an adherens junction protein for cell-cell adhesion and as a signaling protein. ß-catenin function is dependent on its stability, which is regulated by protein-protein interactions that stabilize ß-catenin or target it for proteasome-mediated degradation. In this study, we show that ß-catenin stability is regulated by intracellular pH (pHi) dynamics, with decreased stability at higher pHi in both mammalian cells and Drosophila melanogaster ß-Catenin degradation requires phosphorylation of N-terminal residues for recognition by the E3 ligase ß-TrCP. While ß-catenin phosphorylation was pH independent, higher pHi induced increased ß-TrCP binding and decreased ß-catenin stability. An evolutionarily conserved histidine in ß-catenin (found in the ß-TrCP DSGIHS destruction motif) is required for pH-dependent binding to ß-TrCP. Expressing a cancer-associated H36R-ß-catenin mutant in the Drosophila eye was sufficient to induce Wnt signaling and produced pronounced tumors not seen with other oncogenic ß-catenin alleles. We identify pHi dynamics as a previously unrecognized regulator of ß-catenin stability, functioning in coincidence with phosphorylation.

Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation.

Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. We report that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). We show that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+-H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. Our findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms.

Increased H⁺ efflux is sufficient to induce dysplasia and necessary for viability with oncogene expression.

Intracellular pH (pHi) dynamics is increasingly recognized as an important regulator of a range of normal and pathological cell behaviors. Notably, increased pHi is now acknowledged as a conserved characteristic of cancers and in cell models is confirmed to increase proliferation and migration as well as limit apoptosis. However, the significance of increased pHi for cancer in vivo remains unresolved. Using Drosophila melanogaster, we show that increased pHi is sufficient to induce dysplasia in the absence of other transforming cues and potentiates growth and invasion with oncogenic Ras. Using a genetically encoded biosensor we also confirm increased pHi in situ. Moreover, in Drosophila models and clonal human mammary cells we show that limiting H(+) efflux with oncogenic Raf or Ras induces acidosis and synthetic lethality. Further, we show lethality in invasive primary tumor cell lines with inhibiting H(+) efflux. Synthetic lethality with reduced H(+) efflux and activated oncogene expression could be exploited therapeutically to restrain cancer progression while limiting off-target effects.

Ratiometric imaging of pH probes.

Measurement of intracellular pH can be readily accomplished using tools and methods described in this chapter. We present a discussion of technical considerations of various ratiometric pH-sensitive probes including dyes and genetically encoded sensors. These probes can be used to measure pH across physical scales from macroscopic whole-mount tissues down to organelles and subcellular domains. We describe protocols for loading pH-sensitive probes into single cells or tissues and discuss ratiometric image acquisition and analysis.