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. 2018 Nov 20;25(8):2223-2233.e6.
doi: 10.1016/j.celrep.2018.10.100.

Transmembrane Protease TMPRSS11B Promotes Lung Cancer Growth by Enhancing Lactate Export and Glycolytic Metabolism

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Free PMC article

Transmembrane Protease TMPRSS11B Promotes Lung Cancer Growth by Enhancing Lactate Export and Glycolytic Metabolism

Barrett L Updegraff et al. Cell Rep. .
Free PMC article

Abstract

Pathways underlying metabolic reprogramming in cancer remain incompletely understood. We identify the transmembrane serine protease TMPRSS11B as a gene that promotes transformation of immortalized human bronchial epithelial cells (HBECs). TMPRSS11B is upregulated in human lung squamous cell carcinomas (LSCCs), and high expression is associated with poor survival of non-small cell lung cancer patients. TMPRSS11B inhibition in human LSCCs reduces transformation and tumor growth. Given that TMPRSS11B harbors an extracellular (EC) protease domain, we hypothesized that catalysis of a membrane-bound substrate modulates tumor progression. Interrogation of a set of soluble receptors revealed that TMPRSS11B promotes solubilization of Basigin, an obligate chaperone of the lactate monocarboxylate transporter MCT4. Basigin release mediated by TMPRSS11B enhances lactate export and glycolytic metabolism, thereby promoting tumorigenesis. These findings establish an oncogenic role for TMPRSS11B and provide support for the development of therapies that target this enzyme at the surface of cancer cells.

Keywords: Basigin; CRISPR-mediated genome editing; MCT4; TMPRSS11B; glycolytic metabolism; lactate export; lung cancer; lung squamous cell carcinoma; transmembrane serine protease; transposon mutagenesis.

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. TMPRSS11B Promotes Transformation In Vitro and Tumorigenesis In Vivo
(A) Schematic representation of TMPRSS11B with catalytic residues highlighted. TM, transmembrane; SEA, sea urchin sperm protein, aggrecan, and enterokinase domain; SP, serine protease domain. (B) Soft agar colony formation in human bronchial epithelial cell (HBEC)-shp53 cells expressing GFP (control), D270N and S366A catalytic mutants, or wild-type TMPRSS11B (n = 3, error bars represent SD). (C) Soft agar colony formation in SCC lines expressing mCherry (control) or TMPRSS11B (n = 3, error bars represent SD). (D) Quantitative real-time PCR verification of TMPRSS11B knockdown in cells used for xenograft assays in (E)–(G) (n = 3 for each cell line, error bars represent SD). (E) TMPRSS11B knockdown blunts subcutaneous tumor growth of HCC2814 cells. (Left) Mice treated with doxycycline (dox) water to induce TMPRSS11B knockdown at the time of injection (n = 16 tumors/group). (Right) Mice treated with dox water when tumors were palpable (n = 6 tumors/group). Error bars represent SEM for both right and left graphs. (F) TMPRSS11B knockdown inhibits subcutaneous tumor growth of H157 cells (n = 16 tumors/group, error bars represent SEM). Mice were treated with dox water at the time of injection. (G) TMPRSS11B knockdown reduces subcutaneous tumor growth of DU145 cells (n = 16 tumors/group, error bars represent SEM). Mice were treated with dox water at the time of injection. (H) CRISPR/Cas9 depletion of TMPRSS11B (sgRNA1 clone 3) blunts tumor growth in HCC2814 cells that is restored upon introduction of a CRISPR-resistant TMPRSS11B cDNA (n = 16 tumors/group, error bars represent SEM). Unpaired t test; Benjamini, Krieger, and Yekutieli approach; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 2.
Figure 2.. TMPRSS11B Promotes Basigin Solubilization
(A) Immobilized antibody array on conditioned media harvested from HBEC-shp53-GFP versus HBEC-shp53-TMPRSS11B cells (119 factors profiled, n = 2). (B) Pixel density quantitation of array from (A). Data bars represent SD. (C) Basigin solubilization (conditioned media Basigin:total cellular Basigin) in HBEC-shp53 cells expressing GFP, TMPRSS11B-D270N, TMPRSS11B-S366A, or WT TMPRSS11B (n = 3, error bars represent SD). (D) TMPRSS11B enhances Basigin membrane release in HCC95 and HCC1313 cells (n = 3, error bars represent SD). (E) Inducible shRNA depletion of TMPRSS11B reduces Basigin membrane release in HCC2814 cells (n = 3, error bars represent SD). (F) Treatment of HBEC-shp53 cells with the serine protease inhibitor AEBSF reduces Basigin solubilization (n = 2, error bars represent SD). (G) Treatment of HCC1588, H2073, and HCC2814T (T denotes TMPRSS11B overexpression) with AEBSF reduces Basigin membrane release (n = 3, error bars represent SD). Unpaired t test; Benjamini, Krieger, and Yekutieli approach; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 3.
Figure 3.. TMPRSS11B Interacts and Co-localizes with Basigin and MCT4 at the Plasma Membrane
(A) Reciprocal co-immunoprecipitation (coIP) of HA-tagged TMPRSS11B and V5-tagged Basigin in HBEC-shp53 cells. (B) Reciprocal coIP of V5-tagged TMPRSS11B and an antibody that recognizes endogenous MCT4 in HBEC-shp53 cells. (C) CoIP of endogenous Basigin with three independent antibodies reveals interaction with V5-tagged TMPRSS11B. (D) Immunofluorescence (IF) confocal microscopy of HBEC-shp53-V5-TMPRSS11B cells stained with anti-V5 and anti-Basigin. (E) IF confocal microscopy of HBEC-shp53-V5-TMPRSS11B cells stained with anti-V5 and anti-MCT4. All slides were mounted in solution containing DAPI.
Figure 4.
Figure 4.. TMPRSS11B Regulates Glycolysis and Lactate Export by Enhancing Basigin/ MCT4 Function
(A) Proliferation of isogenic HBEC-shp53 Basigin knockout (KO) clones or sgNS (control non-targeting sgRNA) with either stable GFP or TMPRSS11B expression. (B) Soft agar quantitation of large colonies in HBEC-shp53-GFP and HBEC-shp53-TMPRSS11B with and without Basigin KO (n = 3, error bars represent SD). (C) Intracellular lactate quantification in HBEC-shp53 cells reveals a protease- and Basigin-dependent reduction in cellular lactate content (n = 2, error bars represent SD). (D) SeaHorse analysis of the extracellular acidifica-tion rate (ECAR) in 1.5 3 104 HBEC-shp53 cells reveals enhanced lactate export in HBEC-shp53-TMPRSS11B relative to HBEC-shp53-GFP cells and HBEC-shp53-TMPRSS11B with Basigin knockout (n = 5–6, error bars represent SEM). (E) Relative glycolytic ECAR ((ECAR after glucose injection basal ECAR) normalized to HBEC-shp53-sgNS-GFP) of Basigin KO HBEC-shp53 cells expressing GFP or TMPRSS11B (n = 36 for control sgNS-GFP, n = 72 for experimental groups, error bars represent SEM). (F) Relative ECAR of MCT4 KO and MCT1 inhibitor-treated HBEC-shp53-TMPRSS11B cells (n = 15–18, error bars represent SEM). Unpaired t test; Benjamini, Krieger, and Yekutieli approach; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 5.
Figure 5.. TMPRSS11B Regulates Cellular actate Levels
(A–F) Inducible knockdown of TMPRSS11B pro-motes lactate accumulation in HCC2814 (A), H157 (B), HCC95 (C), and DU145 (D) cells. CRISPR/Cas9 depletion of TMPRSS11B results in lactate accu-mulation in H2073 (E) and HCC2814 (F) cells (n = 3, error bars represent SD). (G) Lactate accumulation in CRISPR/Cas9-modi-fied HCC2814 cells can be rescued by introduction of a CRISPR-resistant TMPRSS11B cDNA (n = 3, error bars represent SD). Unpaired t test; Benjamini, Krieger, and Yekutieli approach; **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 6.
Figure 6.. TMPRSS11B Expression Is Associated with Lactate Secretion in LSCCs
TMPRSS11B mRNA expression correlates with lactate secretion in human LSCCs (n = 12 cell lines, linear regression and goodness of fit analysis).
Figure 7.
Figure 7.. Model of TMPRSS11B-Mediated Regulation of Lactate Export and Glycolysis
(Left) In healthy lung lacking TMPRSS11B expression, Basigin serves a chaperone role in regulating the efficiency of lactate export through MCT4. (Right) In LSCCs with upregulated expression of TMPRSS11B, solubilization of Basigin enhances lactate export efficiency through MCT4, in turn removing a feedback-regulatory role of cellular lactate on glycolytic flux.

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