Acid-base transporters and pH dynamics in human breast carcinomas predict proliferative activity, metastasis, and survival
- Department of Biomedicine, Aarhus University, Denmark
- Department of Pathology, Regionshospitalet Randers, Denmark
- Department of Surgery, Regionshospitalet Randers, Denmark
- Department of Clinical Genetics, University of Southern Denmark, Denmark
- Clinical Genome Center, University and Region of Southern Denmark, Denmark
- Department of Plastic and Breast Surgery, Department of Clinical Medicine, Aarhus University Hospital, Denmark
Figures
Figure 1 with 1 supplement
Cellular net acid extrusion in human breast cancer tissue and normal breast tissue relies on extracellular Na+ and is partially CO2/HCO3–-dependent consistent with the expression of Na+,HCO3– cotransporter NBCn1 and Na+/H+ exchanger NHE1.
Moreover, steady-state intracellular pH (pHi) and the capacity for net acid extrusion are elevated in human breast cancer tissue compared to normal breast tissue. (A) Exemplar fluorescence images of …
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Figure 1—source data 1
Data file containing NBCn1 and NHE1 protein expression levels, steady-state intracellular pH (pHi) values, and net acid extrusion capacities linked to de-identified clinical and pathological patient characteristics.
Note that this source data file pertains to Figure 1 but it also contains the clinicopathological information used for stratification in Figures 2–5, 7, and 8, and Figure 2—figure supplement 1; for the multiple linear and logistic regression analyses in Figure 6; and for the plots in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/68447/elife-68447-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Cellular net acid extrusion activities in presence and nominal absence of CO2/HCO3– plotted as functions of intracellular pH (pHi) for human breast cancer tissue and normal breast tissue.
(A) Primary breast cancer tissue (n=75–76). (B) Normal breast tissue (n=48–49). Figure 1—figure supplement 1—source data 1 provides the data pertaining to this figure and used for stratification in …
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Figure 1—figure supplement 1—source data 1
Data file containing net acid extrusion capacities calculated at specified intracellular pH (pHi) levels and linked to de-identified clinical and pathological patient characteristics.
Note that this source data file pertains to Figure 1—figure supplement 1 but it also contains the clinicopathological information used for stratification in Figure 2—figure supplements 1 and 2, Figure 3—figure supplement 1, Figure 4—figure supplement 1, Figure 5—figure supplement 1, Figure 7—figure supplement 1, and Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/68447/elife-68447-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 2 supplements
Na+,HCO3– cotransport is critical for cellular net acid extrusion and steady-state intracellular pH (pHi) in human invasive ductal and, particularly, lobular breast carcinomas.
(A,B) Traces of NH4+-prepulse-induced pHi dynamics in human invasive lobular (A, n=8–9) and ductal (B, n=60–62) breast carcinomas. The time scale within the dotted rectangles is expanded in order to …
Figure 2—figure supplement 1
Na+/H+ exchange and Na+,HCO3– cotransport activity regulate intracellular pH (pHi) in mucinous adenocarcinomas.
(A) Traces of NH4+-prepulse-induced pHi dynamics in human mucinous adenocarcinomas (n=5). The time scale within the dotted rectangle is expanded in order to improve resolution during the pHi …
Figure 2—figure supplement 2
Cellular net acid extrusion activities in presence and nominal absence of CO2/HCO3– plotted as functions of intracellular pH (pHi) for human invasive lobular and ductal breast carcinomas.
(A) Invasive lobular primary breast carcinomas (n=8–9). (B) Invasive ductal primary breast carcinomas (n=60–62).
Figure 3 with 1 supplement
Steady-state intracellular pH (pHi) is elevated in human breast carcinomas of high malignancy grade due to cellular CO2/HCO3–-dependent net acid extrusion.
(A–C) Traces of NH4+-prepulse-induced pHi dynamics in human invasive ductal breast carcinomas of malignancy grades I (A, n=12–14), II (B, n=34), and III (C, n=14). The time scale within the dotted …
Figure 3—figure supplement 1
Cellular net acid extrusion activities in presence and nominal absence of CO2/HCO3– plotted as functions of intracellular pH (pHi) for human invasive ductal breast carcinomas of malignancy grades I, II, and III.
(A) Primary breast carcinomas of malignancy grade I (n=12–14). (B) Primary breast carcinomas of malignancy grade II (n=34). (C) Primary breast carcinomas of malignancy grade III (n=14).
Figure 4 with 1 supplement
Intracellular pH (pHi) is elevated in estrogen receptor-negative breast cancer.
(A,B) Traces of NH4+-prepulse-induced pHi dynamics in human breast carcinomas stratified by estrogen receptor status (A: negative, n=6; B: positive, n=63–64). The time scale within the dotted …
Figure 4—figure supplement 1
Cellular net acid extrusion activities in presence and nominal absence of CO2/HCO3– plotted as function of intracellular pH (pHi) for human breast carcinomas stratified by estrogen receptor status.
(A) Estrogen receptor-negative primary breast carcinomas (n=6). (B) Estrogen receptor-positive primary breast carcinomas (n=63–64).
Figure 5 with 1 supplement
Protein expression of NHE1 and NBCn1 is elevated in HER2-positive human breast cancer.
(A,B) Traces of NH4+-prepulse-induced intracellular pH (pHi) dynamics in human HER2-positive (A, n=9–11) and HER2-normal (B, n=58–61) breast carcinomas. The time scale within the dotted rectangles …
Figure 5—figure supplement 1
Cellular net acid extrusion activities in the presence and nominal absence of CO2/HCO3– plotted as functions of intracellular pH (pHi) for human breast carcinomas stratified by HER2 status.
(A) HER2-positive primary breast carcinomas (n=9–11). (B) HER2-normal primary breast carcinomas (n=58–61).
Figure 6 with 1 supplement
Histology, malignancy grade, and receptor expression profiles are independent predictors of intracellular pH (pHi) dynamics and acid-base transporter expression in human invasive lobular and ductal breast carcinomas.
(A–D) Multiple linear regression analyses show the independent influences of patient age, tumor size, histology, malignancy grade, and expression of estrogen and HER2 receptors on steady-state pHi …
Figure 6—figure supplement 1
Plots of acid-base parameters in human breast cancer tissue (n=64–74) as functions of tumor size (A,C) and patient age (B,D).
Statistical relationships among the variables were tested by Spearman’s correlation analyses.
Figure 7 with 1 supplement
Steady-state intracellular pH (pHi) is elevated in human breast carcinomas with high proliferative activity (elevated Ki67 index).
(A,B) Traces of NH4+-prepulse-induced pHi dynamics in breast carcinomas with high (A, 35–90% Ki67+, n=19–20) and low (B, 0–30% Ki67+, n=49–51) Ki67 index. The time scale within the dotted rectangles …
Figure 7—figure supplement 1
Cellular net acid extrusion activities in presence and nominal absence of CO2/HCO3– plotted as functions of intracellular pH (pHi) for human breast carcinomas stratified by Ki67 index.
(A) Primary breast carcinomas with high proliferative index (35–90% Ki67+ cells; n=19–20). (B) Primary breast carcinomas with low proliferative index (0–30% Ki67+ cells; n=49–51).
Figure 8 with 1 supplement
Primary breast cancer tissue from patients with axillary lymph node metastases shows higher Na+,HCO3– cotransport activity during intracellular acidification, higher NBCn1 expression, and lower NHE1 expression than breast cancer tissue from patients without metastases.
(A,B) Traces of NH4+-prepulse-induced intracellular pH (pHi) dynamics in primary breast carcinomas from patients with (A, n=25–28) and without (B, n=42–44) axillary lymph node metastases. Time …
Figure 8—figure supplement 1
Cellular net acid extrusion activities in presence and nominal absence of CO2/HCO3– plotted as functions of intracellular pH (pHi) for human primary breast carcinomas stratified by axillary lymph node status.
(A) Primary breast carcinomas from patients with lymph node metastases (n=25–28). (B) Primary breast carcinomas from patients with no lymph node metastases (n=42–44).
Figure 9 with 1 supplement
The levels of SLC9A1 mRNA, encoding NHE1, vary among breast cancer subtypes, and high SLC9A1 expression is associated with improved survival in patients with luminal A breast cancer.
(A) Variation in SLC9A1 mRNA levels among patients with different breast cancer subtypes (n=135–344). Expression data were compared by one-way ANOVA followed by Tukey’s post-test. Figure 9—figure …
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Figure 9—source data 1
The mRNA expression for SLC9A1 correlates with that of ESR1 and ERBB2.
When adjusted for expression of ESR1, PGR, and ERBB2, the mRNA expression of SLC9A1 does not correlate with that of SLC4A7, SLC16A1, and SLC16A3 (n=409–1162).
- https://cdn.elifesciences.org/articles/68447/elife-68447-fig9-data1-v2.docx
Figure 9—figure supplement 1
Transcript patterns for the different molecular subtypes of breast cancer.
We observe systematic differences in mRNA levels for estrogen (A: ESR1, n=135–344), progesterone (B: PGR, n=162–438), and HER2 (C: ERBB2, n=162–438) receptors, the proliferative marker Ki67 (D: MKI67…
Figure 10 with 2 supplements
The levels of SLC4A7 mRNA, encoding NBCn1, vary among breast cancer subtypes, and high SLC4A7 expression is associated with poor survival in patients with luminal A or basal-like breast cancer.
(A) Variation in SLC4A7 mRNA levels among patients with different breast cancer subtypes (n=135–344). Expression data were compared by one-way ANOVA followed by Tukey’s post-test. *p<0.05, ***p<0.001…
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Figure 10—source data 1
The mRNA expression for SLC4A7 correlates with that of ESR1, PGR, and ERBB2.
When adjusted for expression of ESR1, PGR, and ERBB2, the mRNA expression of SLC4A7 does not correlate with that of SLC9A1, SLC16A1, and SLC16A3 (n=409–1162).
- https://cdn.elifesciences.org/articles/68447/elife-68447-fig10-data1-v2.docx
Figure 10—figure supplement 1
The levels of SLC16A1 mRNA, encoding MCT1, vary among breast cancer subtypes but are not associated with significant changes in patient survival.
(A) Variation in SLC16A1 mRNA levels among patients with different breast cancer subtypes (n=162–438). Expression data were compared by one-way ANOVA followed by Tukey’s post-test. Figure 10—figure …
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Figure 10—figure supplement 1—source data 1
The mRNA expression for SLC16A1 correlates with that of ESR1, PGR, and ERBB2.
When adjusted for expression of ESR1, PGR, and ERBB2, the mRNA expression of SLC16A1 does not correlate with that of SLC4A7, SLC9A1, and SLC16A3 (n=409–1457).
- https://cdn.elifesciences.org/articles/68447/elife-68447-fig10-figsupp1-data1-v2.docx
Figure 10—figure supplement 2
The levels of SLC16A3 mRNA, encoding MCT4, vary among breast cancer subtypes, and high SLC16A3 expression is associated with poor survival except in luminal A breast cancer.
(A) Variation in SLC16A3 mRNA levels among patients with different breast cancer subtypes (24–141). Expression data were compared by one-way ANOVA followed by Tukey’s post-test. Figure 10—figure …
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Figure 10—figure supplement 2—source data 1
The mRNA expression for SLC16A3 correlates with that of ESR1, PGR, and ERBB2.
When adjusted for expression of ESR1, PGR, and ERBB2, the mRNA expression of SLC16A3 does not correlate with that of SLC4A7, SLC9A1, and SLC16A1 (n=409).
- https://cdn.elifesciences.org/articles/68447/elife-68447-fig10-figsupp2-data1-v2.docx
Figure 11
Schematics summarizing identified interactions and associated functional implications of NHE1 (upper panel) and NBCn1 (lower panel) in human breast cancer.
The image was generated with BioRender.com.
Tables
Table 1
Clinical and pathological characteristics of the patient cohort investigated for intracellular pH (pHi) dynamics and protein expression.
| Number of patients | 110 |
| Patient age (years; median, interquartile range) | 64.5 (56–74) |
| Tumor size (mm; median, interquartile range) | 18 (14–26) |
| Histological type | |
| Invasive ductal carcinomas | 92 (84%) |
| Invasive lobular carcinomas | 92 (84%) |
| Mucinous adenocarcinomas | 5 (5%) |
| Tubular carcinoma | 2 (2%) |
| Pleomorphic lobular carcinoma | 1 (1%) |
| HER2 receptor status | |
| Normal | 95 (86%) |
| Overexpression or gene amplification | 15 (14%) |
| Estrogen receptor status | |
| 90–100% ER+ cells | 99 (90%) |
| 0–15% ER+ cells | 11 (10%) |
| Malignancy grade | |
| I | 31 (28%) |
| II | 52 (47%) |
| III | 22 (20%) |
| Not graded | 5 (5%) |
| Axillary lymph node status | |
| Negative | 69 (63%) |
| Isolated tumor cells | 15 (14%) |
| Micro-metastases | 5 (5%) |
| Macro-metastases | 21 (19%) |
| Ki67 index | |
| 0–30% Ki67+ cells | 82 (75%) |
| 35–90% Ki67+ cells | 28 (25%) |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | SLC4A7 | GenBank | Gene ID: 9497 | Encodes NBCn1 |
| Gene (Homo sapiens) | SLC9A1 | GenBank | Gene ID: 6548 | Encodes NHE1 |
| Gene (Homo sapiens) | SLC16A1 | GenBank | Gene ID: 6566 | Encodes MCT1 |
| Gene (Homo sapiens) | SLC16A3 | GenBank | Gene ID: 9123 | Encodes MCT4 |
| Biological sample (Homo sapiens) | Surgical breast biopsies | Regionshospitalet Randers, Denmark | This study cohort | Cancer and matched normal tissue |
| Chemical compound, drug | Collagenase type 3 | Worthington Biochemical Corporation | Cat. #: LS004182 | 450 IU/mL |
| Chemical compound, drug | BCECF-AM | Thermo Fisher Scientific | Cat. #: B1170 | 3 µM |
| Antibody | Anti-NBCn1 (Rabbit polyclonal) | Jeppe Praetorius, Aarhus University, Denmark Damkier et al., 2006 | Reference | IHC (1:100) |
| Antibody | Anti-NHE1 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat. #: sc-136239; RRID:AB_2191254 | IHC (1:100) |
| Commercial assay, kit | OptiView DAB IHC detection kit | Roche Diagnostics | RRID:AB_2833075 | Goat anti-rabbit and anti-mouse |
| Software, algorithm | SPSS | IBM | RRID:SCR_002865 | |
| Software, algorithm | Prism | GraphPad | RRID:SCR_002798 | Version 9.1.1 |
