Mechanosensitive pannexin-1 channels mediate microvascular metastatic cell survival

Journal name:
Nature Cell Biology
Volume:
17,
Pages:
943–952
Year published:
DOI:
doi:10.1038/ncb3194
Received
Accepted
Published online

Abstract

During metastatic progression, circulating cancer cells become lodged within the microvasculature of end organs, where most die from mechanical deformation. Although this phenomenon was first described over a half-century ago, the mechanisms enabling certain cells to survive this metastasis-suppressive barrier remain unknown. By applying whole-transcriptome RNA-sequencing technology to isogenic cancer cells of differing metastatic capacities, we identified a mutation encoding a truncated form of the pannexin-1 (PANX1) channel, PANX11–89, as recurrently enriched in highly metastatic breast cancer cells. PANX11–89 functions to permit metastatic cell survival during traumatic deformation in the microvasculature by augmenting ATP release from mechanosensitive PANX1 channels activated by membrane stretch. PANX1-mediated ATP release acts as an autocrine suppressor of deformation-induced apoptosis through P2Y-purinergic receptors. Finally, small-molecule therapeutic inhibition of PANX1 channels is found to reduce the efficiency of breast cancer metastasis. These data suggest a molecular basis for metastatic cell survival on microvasculature-induced biomechanical trauma.

At a glance

Figures

  1. PANX11-89 augments PANX1-mediated ATP release in metastatic breast cancer cells.
    Figure 1: PANX11–89 augments PANX1-mediated ATP release in metastatic breast cancer cells.

    (a) Sanger sequencing traces from the cDNA of CN34, CN-LM1A, MDA-MB-231 (abbreviated to MDA on the figure) and MDA-LM2 cells at the nSNV alleles predicted to result in non-neutral substitutions by PolyPhen-2. (b) Quantification of PANX1-mediated ATP release from CN-LM1A cells pretreated for 10 min with PBS, 2 mM probenecid, 500 μM CBX, or 100 μM 10Panx1 peptide; n = 4. (c) Quantification of PANX1-mediated ATP release from HEK293T cells transfected with 8 μg control vector (n = 8), 8 μg wild-type PANX1 (n = 8), 5 μg wild-type PANX1 and 3 μg PANX11–89 (n = 7), or 8 μg PANX11–89 (n = 3), and pretreated for 10 min with 500 μM CBX or an equivalent volume of PBS. (d) Quantification of ATP release from PANX11–89-expressing HCC1806 breast cancer cells transfected with siRNAs against full-length endogenous PANX1 or control siRNA; n = 12. (e) Time-course measurements of CBX-sensitive ATP release from CN34 parental cells and the CN-LM1A metastatic derivative sub-line pretreated with CBX (500 μM) or PBS for 10 min; n = 4. Error bars, s.e.m., NS, not significant; , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents biological replicates. Experimental results presented are representative and were independently replicated at least two times with two independent cell lines.

  2. Breast cancer cell PANX1 activity within the pulmonary vasculature promotes lung metastasis.
    Figure 2: Breast cancer cell PANX1 activity within the pulmonary vasculature promotes lung metastasis.

    (a) Quantitative bioluminescence imaging of extracellular ATP release by cancer cells in the lung vasculature 5 min after tail-vein injection of 1 × 105 MDA-LM2 cells expressing plasma-membrane-anchored extracellular luciferase (MDA-LM2-pmeLUC). MDA-LM2-pmeLUC cells were pretreated for 10 min with either CBX (500 μM; n = 5) or PBS (n = 7) before injection into FVB/NJ mice. (b) Quantitative bioluminescence imaging of lung metastasis after the injection of 1 × 105 highly metastatic CN-LM1A breast cancer cells pretreated with 100 μM 10Panx1 (n = 6) or scrambled peptide (n = 7), into NOD scid mice. (c) Day 42 quantification of metastatic foci from haematoxylin and eosin (H&E)-stained lungs (left) and representative images from vimentin-stained lungs (right) of mice injected with CN-LM1A cells pretreated with 10Panx1 or scrambled peptide; n = 5. Scale bar, 0.5 mm. (d) Daily quantitative imaging plot of lung bioluminescence subsequent to the injection of 1 × 105 metastatic CN-LM1A breast cancer cells pretreated (30 min) with 100 μM 10Panx1 or scrambled peptides, into NOD scid mice; n = 7. (e) Lungs from mice were extracted at day 3, sectioned and stained for vimentin and the numbers of vimentin-positive cancer cells were quantified; n = 5. Scale bar, 0.25 mm. (fIn vivo quantification of luciferase-based caspase-3/7 activity at 3 and 6 h after tail-vein injection of 1 × 105 CN-LM1A breast cancer cells, pretreated with 100 μM 10Panx1 or scrambled peptide, into NOD scid mice; n = 5. (g) Quantitative bioluminescence imaging of cancer cells in the lungs 8 h after the injection of 2 × 105 BT549 cells transfected with siRNAs against PANX1 or control siRNA; n = 5. Error bars, s.e.m., , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents number of mice in a,b,d,f,g and mouse lungs in c and e. Experiments bf are representative and were replicated at least two times in independent cell lines. Bioluminescent and histological images are representative of the median.

  3. Mechanosensitive PANX1 channels release ATP to increase cancer cell survival during intravascular membrane stretch.
    Figure 3: Mechanosensitive PANX1 channels release ATP to increase cancer cell survival during intravascular membrane stretch.

    (a) Representative images of mouse lungs stained for cancer cells and blood vessels (green and magenta, respectively, top panel), blood vessels (black, middle panel) or cleaved caspase-3 (white, bottom panel) 3 h after tail-vein injection of 1 × 105 CN-LM1A cells. Arrows indicate endothelium. Scale bars, 10 μm. (b) Quantification of PANX1-mediated CBX-sensitive ATP release from CN-LM1A and MDA-LM2 cells during 5 min exposure to isotonic (100% PBS) or hypotonic (70% PBS) solution; n = 4 (CN-LM1A isotonic), n = 3 (CN-LM1A hypotonic), n = 4 (MDA-LM2 isotonic), n = 4 (MDA-LM2). (c) Quantification of viable, trypan blue-negative, CN34 parental and CN-LM1A derivative cells after 1 h extreme hypotonic (12.5% PBS) stretch; n = 4. (d) Quantification of viable, trypan blue-negative MDA-MB-231 parental and MDA-LM2 derivative cells after 1 h extreme hypotonic (12.5% PBS) stretch; n = 4. (e) Quantification of viable, trypan blue-negative, CN-LM1A cells after 1 h incubation in extremely hypotonic (12.5% PBS) solution in the presence of scrambled peptide (100 μM), 10Panx1 peptide (100 μM) or 10Panx1 peptide (100 μM) and 100 μM ATP; n = 4. (f) Quantification of viable, trypan blue-negative, MDA-LM2 cells after 1 h incubation in extremely hypotonic (12.5% PBS) solution in the presence of scrambled peptide (100 μM), 10Panx1 peptide (100 μM) or 10Panx1 peptide (100 μM) and 100 μM ATP; n = 4. (g) Quantification of viable, trypan blue-negative MDA-LM2 cells with 30 min Boyden chamber centrifugation (1,243g) after cells were pretreated for 10 min with 500 μM CBX or PBS; n = 4. Error bars, s.e.m., , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents biological replicates. Experiments bg are representative and were replicated at least two times with two independent cell lines.

  4. Extracellular ATP enhances metastatic survival through breast cancer cell-autonomous purinergic signalling.
    Figure 4: Extracellular ATP enhances metastatic survival through breast cancer cell-autonomous purinergic signalling.

    (a) Quantitative bioluminescence imaging of lung metastasis after tail-vein injection of 1 × 106 metastatic CN-LM1A cells, expressing CD39 or control vector, into NOD scid mice; n = 12. (b) Lungs from day 42 were extracted, H&E stained, and the numbers of metastatic foci were quantified; n = 12. Scale bar, 1 mm. (c) Quantitative imaging of lung bioluminescence at 6 h post tail-vein injection of 1 × 105 CN-LM1A breast cancer cells pretreated (30 min) and co-injected with apyrase (2 U ml−1) into FVB/NJ mice; n = 6. (d) Quantitative imaging of lung bioluminescence at 6 h post tail-vein injection of 4 × 104 MDA-LM2 breast cancer cells pretreated (30 min) and co-injected with apyrase (2 U ml−1) into FVB/NJ mice; n = 6. (e) Quantification of viable, trypan blue-negative, CN-LM1A cells after 15 min incubation in extremely hypotonic (12.5% PBS) solution in the presence of suramin (50 μM) or water vehicle; n = 4. (f) Quantification of viable, trypan blue-negative MDA-LM2 cells after 15 min incubation in extremely hypotonic (12.5% PBS) solution in the presence of suramin (50 μM) or water vehicle; n = 4. (g) Confocal microscopy images of HEK293T cells expressing PANX1–EGFP (green) and PANX11–89–mRFP (magenta). Co-localization at the plasma membrane is shown by channel overlay (white). Nuclear (nuc) DAPI stain is indicated. Arrowheads indicate PANX1–EGFP and PANX11–89–mRFP1 co-localization at the plasma membrane. Scale bar, 5 μm. (h) Co-immunoprecipitation of PANX11–89–RFP from protein-crosslinked (2 mM dithiobis(succinimidyl propionate)) HEK293T cells expressing PANX1–EGFP, PANX1–EGFP and PANX11–89–mRFP, or PANX11–89–mRFP. Anti-GFP antibody was used to detect the wild-type PANX1 in complex with mutant PANX11–89. Molecular weights are indicated. (i) Quantification of ATP release from HEK293T cells transfected with 5 μg wild-type PANX1 (n = 8), 5 μg wild-type PANX1 and 2.5 μg PANX11–89 (n = 9), 5 μg C-terminus-deleted PANX11–297 (n = 11) or 5 μg PANX11–297 and 2.5 μg PANX11–89 (n = 12). Error bars, s.e.m., NS, not significant, , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents number of mice in a,c,d, number of mouse lungs in b, and biological replicates in e,f,i. Experiments cf are representative and were replicated at least two times with independent cell lines. Bioluminescent and histological images are representative of the median. Unprocessed original scans of blots are shown in Supplementary Fig. 6.

  5. Mutational augmentation of PANX1 channel activity enhances the metastatic efficiency of breast cancer cells.
    Figure 5: Mutational augmentation of PANX1 channel activity enhances the metastatic efficiency of breast cancer cells.

    (a) The numbers of vimentin-positive breast cancer cells in the lung were counted one week after the extraction of size-matched mammary fat pad primary tumours generated by the orthotopic injection of 2.5 × 105 MDA-MB-468 cells expressing PANX11–89 or control vector into NOD scid gamma (NSG) mice; n = 4. Scale bar, 100 μm. (b) Quantitative bioluminescence imaging of systemic metastasis one week after the extraction of size-matched mammary fat pad tumours generated by the orthotopic injection of 5 × 105 HCC1806 breast cancer cells expressing PANX11–89 (n = 7) or control vector (n = 9) into NSG mice. (c) Quantitative bioluminescence imaging of systemic metastasis after tail-vein injection of 1 × 106 MDA-MB-468 breast cancer cells, expressing PANX11–89 or control vector, into NSG mice; n = 4. (d) Ex vivo bioluminescence imaging of metastatic target organs (lung, liver and bone) 14 days after tail-vein injection of MDA-MB-468 cells. (e) Quantitative imaging of lung bioluminescence 18 h post tail-vein injection of 1 × 106 BT549 cells, expressing PANX11–89 or a control vector, into NSG mice; n = 6. (f) In vivo quantification of luciferase-based caspase-3/7 activity at 3 and 6 h post tail-vein injection of 1 × 106 BT549 cells, expressing either PANX11–89 or a control vector, into NOD scid mice; n = 4 (3 h control), n = 5 (3 h PANX11–89), n = 4 (6 h control), n = 5 (6 h PANX11–89). (g) Quantification of viable, trypan blue-negative BT549 cells expressing PANX11–89 or a control vector after 1 h extreme hypotonic (12.5% PBS) stretch in the presence of succinate buffer or apyrase (2 U ml−1); n = 3. Error bars, s.e.m., , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents number of mouse lungs in a, number of mice in b,c,e,f, and biological replicates in g. Orthotopic experiments were replicated in two independent triple-negative cell human breast cancer cell lines. Experiments eg are representative and were replicated at least two times in at least two independent cell lines. Bioluminescent and histological images are representative of the median.

  6. Pharmacological PANX1 channel blockade reduces breast cancer metastasis to the lungs.
    Figure 6: Pharmacological PANX1 channel blockade reduces breast cancer metastasis to the lungs.

    (a) Schematic depicting the two in vivo CBX therapy regimens tested. (b) Quantitative bioluminescence imaging of lung metastasis after tail-vein injection of 5 × 104 MDA-LM2 breast cancer cells into NOD scid mice pretreated daily with 25 mg kg−1 i.p. CBX or an equivalent volume of PBS for six days and with 100 mg kg−1 i.v. CBX or an equivalent volume of PBS 30 min before cancer cell injection; n = 8 (vehicle), n = 9 (CBX). (c) Quantification of the number of metastatic foci at 2 weeks from H&E-stained lungs and representative images from vimentin-stained lungs; n = 4. Scale bar, 0.5 mm. (d) Quantitative bioluminescence imaging of lungs 24 h after tail-vein injection of 1 × 105 CN-LM1A breast cancer cells into NOD scid mice pretreated with 25 mg kg−1 i.p. CBX or an equivalent volume of PBS at 19 and 2 h before cancer cell injection; n = 10. (e) Lungs were extracted at 24 h, sectioned and stained for vimentin and the number of vimentin-positive cancer cells was quantified; n = 8 (vehicle), n = 7 (CBX). Scale bar, 0.25 mm. (f) Quantitative bioluminescence imaging of breast cancer cells in the lungs at 4 weeks after tail-vein injection of 1 × 105 CN-LM1A breast cancer cells into NOD scid mice pretreated with 25 mg kg−1 i.p. CBX (n = 9) or an equivalent volume of PBS (n = 10) at 19 and 2 h before cancer cell injection. (g) Quantification of the number of metastatic foci at 4 weeks from H&E-stained lungs; n = 10 (vehicle), n = 9 (CBX). Error bars, s.e.m., , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents number of mice in b,d,f, number of mouse lungs in c,e,g. Therapeutic PANX1 inhibition experiments were performed using two independent triple-negative breast cancer cell lines. Bioluminescent and histological images are representative of the median.

  7. Proposed working model of PANX1-mediated ATP release as a regulator of intravascular metastatic cell survival.
    Figure 7: Proposed working model of PANX1-mediated ATP release as a regulator of intravascular metastatic cell survival.

    Schematic showing stretch-induced ATP release through mechanosensitive PANX1 channels activating a cancer cell-autonomous purinergic signalling pathway that inhibits cell death during mechanical stress in the microvasculature of target organs. Cancer cells enabled to secrete the levels of extracellular ATP necessary to permit intravascular survival through the activation of autocrine purinergic signalling pathways, or the adjacent cells lodged within the same tumour cell cluster, maintain an opportunity to undergo the subsequent steps of the metastatic cascade—extravasation, colonization, re-initiation and proliferation.

  8. The discovery of recurrent mutations enriched in highly metastatic breast cancer.
    Supplementary Fig. 1: The discovery of recurrent mutations enriched in highly metastatic breast cancer.

    (a) Schematic of the systematic discovery framework used to identify nSNVs enriched by allelic frequency in highly metastatic CN-LM1A and MDA-LM2 human breast cancer cells. (b) Recurrent and non-neutral mutations identified to be significantly enriched in highly metastatic breast cancer cells by a one-tailed Students t-test (P < 0.05); n = 4. (c) Allele-specific RNA-seq of the PANX1 C268T allele in biological triplicates of the CN34 and MDA-MB-231 parental breast cancer cells and their respective lung metastatic derivatives, CN-LM1A and MDA-LM2. Mean enrichment was quantified by counting the increase in frequency of the PANX1 C268T allele in the metastatic sub-lines as compared to the corresponding parental lines; n = 3. (d) Sanger sequencing of the PANX1 mutant allele from genomic DNA of each parental and metastatic line. (e) Metaphase spread of four single-cell subclones generated from the MDA-LM2 cell population showing the intraclonal genetic heterogeneity (varying number of chromosomes) of in vivo selected highly metastatic derivative cell lines. Fractional enrichment of nSNVs in metastatic derivatives can be understood in the context of such heterogeneity. n represents biological replicates. Experiments bd are representative and were replicated with two independent metastatic breast cancer cell lines.

  9. PANX11-89 augments PANX1 channel-mediated extracellular ATP release from metastatic breast cancer cells.
    Supplementary Fig. 2: PANX11–89 augments PANX1 channel-mediated extracellular ATP release from metastatic breast cancer cells.

    (a) The % inhibition of extracellular ATP release from metastatic derivative sub-lines at one minute was measured in the presence of three independent PANX1 inhibitors (Probenecid, Cbx and 10Panx1) at varying concentrations (2 mM, 500 μM and 100 μM, respectively). (b) Increasing concentrations of ATP (0, 50, 100, 500 nM) were measured using the Cell-Titer Glo luciferase assay. (c) Quantification of PANX1-mediated ATP release from the MDA-LM2 sub-line pretreated for 10 min with PBS, 2 mM probenecid (Prob), 500 μM CBX, or 100 μM 10Panx1 peptide; n = 4. (d) Extracellular ATP release from PANX1-null mouse embryonic fibroblasts (PANX1 KO MEFs) transfected with 5 μg human full-length PANX1 or 5 μg human full-length PANX1 and 2.5 μg human PANX11–89; n = 7. (e) Extracellular ATP release from PANX1 KO MEFs transfected with 5 μg human PANX11–89 or 5 μg vector control; n = 8. (f) PANX1-mediated extracellular ATP release from BT549 breast cancer cells expressing PANX11–89 or control vector; n = 8. (g) PANX1-mediated extracellular ATP release from MDA-MB-468 breast cancer cells expressing PANX11–89 or control vector; n = 8. (h) Quantitative RT-PCR analysis of wild-type PANX1 transcript expression in HCC1806 cells expressing PANX11–89 transfected with two independent siRNAs that specifically target full-length PANX1; n = 4. (i) CN34 and CN-LM1A total PANX1 mRNA expression quantified by RNA-seq; FPKM values averaged over two rounds (4 technical replicates) of RNA-seq for each cell line; n = 2. (j) Time-course measurements of ATP release from MDA-MB-231 parental cells and the MDA-LM2 metastatic derivatives sub-lines pretreated with Cbx (500 μM) or PBS for 10 min; n = 4. (k) Quantitative bioluminescence imaging of lung metastasis after the injection of 4 × 104 MDA-LM2 breast cancer cells pretreated with 100 μM 10Panx1 or scrambled peptide, into NOD scid (NS) mice; n = 5. (l) Day 42 quantification of metastatic foci from H&E-stained lungs (left) and representative lung images from vimentin-stained lungs (right) of mice injected with MDA-LM2 cells pretreated with 10Panx1 or scrambled peptide; n = 5. Scale bar, 1 mm. Error bars, s.e.m., ns, nonsignificant, , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents biological replicates ch,l,j, number of mice in k and number of lungs in l. Experiments cg and il were replicated twice with two independent cell lines. Experiments h and i are single experiments with biological replicates. Bioluminescent and histological images are representative of the median.

  10. PANX1 activity promotes the metastatic dissemination of breast cancer cells by enhancing early survival in the target organ.
    Supplementary Fig. 3: PANX1 activity promotes the metastatic dissemination of breast cancer cells by enhancing early survival in the target organ.

    (a) Daily quantitative imaging plot of lung bioluminescence subsequent to the injection of 4 × 104 metastatic MDA-LM2 breast cancer cells pretreated (30 min) with 100 μM 10Panx1 or scrambled peptides, into NS mice; n = 7. (b) Lungs from mice were extracted at day 3, sectioned and stained for vimentin and the numbers of vimentin-positive cancer cells were quantified; n = 7. Scale bar, 0.25 mm. (c) Quantification of proliferation over 5 days for CN-LM1A and MDA-LM2 cells over-expressing the autoinhibitory C-terminal domain of PANX1 or control vector; n = 4. (d) Quantification of 24 h cell survival for CN-LM1A and MDA-LM2 cells pretreated for 15 min with 100 μM 10Panx1 or scrambled peptide; n = 8. (e) Quantification of 24 h invasion for CN-LM1A and MDA-LM2 cells in the presence of 100 μM 10Panx1 or scrambled peptide; n = 4. (f) Quantification of 36 h anchorage-independent cancer cell survival for CN-LM1A cells in the presence of 100 μM 10Panx1 or scrambled peptide; n = 4. (g) Quantification of 24 h trans-endothelial migration for MDA-LM2 cells in the presence of 100 μM 10Panx1 or scrambled peptide; n = 4. (h) In vivo quantification of luciferase-based caspase-3/7 activity at 3 and 6 h after tail-vein injection of 4 × 104 MDA-LM2 breast cancer cells, pretreated with 100 μM 10Panx1 or scrambled peptide, into NS mice; n = 5. Error bars, s.e.m., , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents number of mice in a,h, number of mouse lungs in b, and biological replicates in cg. Experiments ae and h were replicated with at least two times in two independent cell lines. Experiments f and g are single experiments with biological replicates. Bioluminescent and histological images are representative of the median.

  11. PANX11-89 augments ATP release through an interaction with PANX1 to promote breast cancer cell survival in the pulmonary vasculature.
    Supplementary Fig. 4: PANX11–89 augments ATP release through an interaction with PANX1 to promote breast cancer cell survival in the pulmonary vasculature.

    (a) Quantification of viable, trypan blue-negative CN-LM1A cells with 30 min Boyden chamber centrifugation (3,800 r.p.m.) after cells were pre-treated for 10 min with 500 μM CBX or PBS; n = 4. (b) Quantification of extracellular ATP release from CN-LM1A cells expressing the extracellular ATP hydrolase CD39 or control vector; n = 8. (c) Daily quantitative imaging of lung bioluminescence for three days subsequent to the injection of 1 × 105 CN-LM1A breast cancer cells expressing CD39 or control vector, into NS mice; n = 6. (d) Co-immunoprecipiation of Flag-tagged full-length PANX11−426 and endogenous PANX11–89 from CN-LM1A and MDA-LM2 cells. Anti-PANX1 N-terminal antibody detected a band similar in size to that of Flag-tagged PANX11–89 expressed in HEK293T cells. The presence of this band in the metastatic sub-lines suggests that endogenous PANX11–89 associates with Flag-tagged full-length PANX11−426. The multiple bands representing full-length PANX1 represent the previously described glycosylated forms of PANX1. (e) Co-immunoprecipitation of PANX11–89-Flag from HEK293T cells co-transfected with full-length PANX11−426. Anti-PANX1 N-terminal antibody was used to detect the associated PANX1 species. The multiple full-length PANX1 bands represent the previously described glycosylated forms of PANX1. The input lysates were immunoblotted for PANX1. (f) Increasing concentrations of DSP (dithiobis[succinimidyl propionate]) crosslinker were applied to HEK293T cells expressing Flag-tagged PANX1 before lysis. PANX1 complexes were detected using anti-FLAG M2 antibody. (g) Anti-PANX1 and anti-RFP immunoblotting of DSP crosslinked lysates from HEK293T cells expressing PANX1-EGFP, PANX1-EGFP and PANX11–89-mRFP or PANX11–89-mRFP. Molecular weights are indicated. (h) Quantification of PANX1-mediated ATP release from HEK293T cells transfected with 5 μg control vector, 5 μg wild-type PANX1 (DVVD), 2.5 μg wild-type PANX1 and 2.5 μg PANX11–89, 5 μg caspase resistant full-length PANX1 (AVVA), or 2.5 μg caspase resistant full-length PANX1 and 2.5 μg PANX11–89; n = 4. Error bars, s.e.m., , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents biological replicates in a,b,h, and number of mice in c. Experiments ad were replicated at least two times with at least two independent cell lines. Experiments eh are single experiments with biological replicates. Bioluminescent and images are representative of the median.

  12. PANX1 is a druggable target promoting breast cancer metastasis on cancer cell entry into the lung.
    Supplementary Fig. 5: PANX1 is a druggable target promoting breast cancer metastasis on cancer cell entry into the lung.

    (a) Quantitative imaging of lung bioluminescence 18 h post tail-vein injection of 1 × 106 MDA-MB-468 breast cancer cells, expressing PANX11–89 or a control vector, into NSG mice; n = 6. (b) In vivo quantification of luciferase-based caspase-3/7 activity at 3 and 6 h post tail-vein injection of 1 × 106 MDA-MB-468 breast cancer cells, expressing either PANX11–89 or a control vector, into NS mice; n = 6. (c) Quantification of viable, trypan blue-negative MDA-MDA-468 cells expressing PANX11–89 or a control vector after 1 h extreme hypotonic (12.5% PBS) stretch in the presence of succinate buffer or apyrase (2 U ml−1); n = 4. (d) Quantitative bioluminescence imaging of lung metastasis after tail-vein injection of 1 × 105 CN-LM1A breast cancer cells pretreated for 30 min with CBX (500 μM) or PBS vehicle into NS mice; n = 4 (vehicle), n = 6 (Cbx). (e) Day 35 quantification of metastatic foci (left) and representative lung images (right) from H&E stained lungs of mice injected with CN-LM1A cells pretreated with CBX or PBS vehicle; n = 4 (vehicle), n = 6 (Cbx). Scale bar, 0.5 mm. (f) Mouse body weight before and after daily i.p. injections of CBX (25 mg kg−1) or an equivalent volume of PBS vehicle for seven days. n = 10. (g) 4-week representative images of vimentin-stained lungs from mice treated with two 25 mg kg−1 doses of intravenous CBX or vehicle control. Arrowheads indicate vimentin-positive metastatic foci. Scale bar, 0.5 mm. Error bars, s.e.m., ns, nonsignificant; , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents number of mice in a,b,d,f, number of mouse lungs in e and biological replicates in c. Experiments ac were replicated at least two times with at least two independent cell lines. Therapeutic PANX1 inhibition experiments were performed using differing protocols with two independent triple negative breast cancer cell lines. Bioluminescent and histological images are representative of the median.

  13. Unprocessed scans of immunoblots.
    Supplementary Fig. 6: Unprocessed scans of immunoblots.

Tables

  1. RNA-seq profile of CN34, CN-LM1A, MDA-MB-231 and MDA-LM2 cell populations.
    Supplementary Table 1: RNA-seq profile of CN34, CN-LM1A, MDA-MB-231 and MDA-LM2 cell populations.
  2. Primers used in this study.
    Supplementary Table 2: Primers used in this study.

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Author information

Affiliations

  1. Laboratory of Systems Cancer Biology, Rockefeller University, 1230 York Avenue New York, New York 10065, USA

    • Paul W. Furlow,
    • Steven Zhang,
    • Nils Halberg,
    • Hani Goodarzi,
    • Creed Mangrum,
    • Y. Gloria Wu &
    • Sohail F. Tavazoie
  2. HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, New York, New York 10021, USA

    • T. David Soong &
    • Olivier Elemento

Contributions

S.F.T. conceived the project and supervised all research. P.W.F. and S.F.T. wrote the manuscript. P.W.F. and S.F.T. designed the experiments. P.W.F., S.Z., N.H. and C.M. performed the experiments. T.D.S., H.G., P.W.F. and O.E. designed and performed the computational analysis.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: The discovery of recurrent mutations enriched in highly metastatic breast cancer. (171 KB)

    (a) Schematic of the systematic discovery framework used to identify nSNVs enriched by allelic frequency in highly metastatic CN-LM1A and MDA-LM2 human breast cancer cells. (b) Recurrent and non-neutral mutations identified to be significantly enriched in highly metastatic breast cancer cells by a one-tailed Students t-test (P < 0.05); n = 4. (c) Allele-specific RNA-seq of the PANX1 C268T allele in biological triplicates of the CN34 and MDA-MB-231 parental breast cancer cells and their respective lung metastatic derivatives, CN-LM1A and MDA-LM2. Mean enrichment was quantified by counting the increase in frequency of the PANX1 C268T allele in the metastatic sub-lines as compared to the corresponding parental lines; n = 3. (d) Sanger sequencing of the PANX1 mutant allele from genomic DNA of each parental and metastatic line. (e) Metaphase spread of four single-cell subclones generated from the MDA-LM2 cell population showing the intraclonal genetic heterogeneity (varying number of chromosomes) of in vivo selected highly metastatic derivative cell lines. Fractional enrichment of nSNVs in metastatic derivatives can be understood in the context of such heterogeneity. n represents biological replicates. Experiments bd are representative and were replicated with two independent metastatic breast cancer cell lines.

  2. Supplementary Figure 2: PANX11–89 augments PANX1 channel-mediated extracellular ATP release from metastatic breast cancer cells. (323 KB)

    (a) The % inhibition of extracellular ATP release from metastatic derivative sub-lines at one minute was measured in the presence of three independent PANX1 inhibitors (Probenecid, Cbx and 10Panx1) at varying concentrations (2 mM, 500 μM and 100 μM, respectively). (b) Increasing concentrations of ATP (0, 50, 100, 500 nM) were measured using the Cell-Titer Glo luciferase assay. (c) Quantification of PANX1-mediated ATP release from the MDA-LM2 sub-line pretreated for 10 min with PBS, 2 mM probenecid (Prob), 500 μM CBX, or 100 μM 10Panx1 peptide; n = 4. (d) Extracellular ATP release from PANX1-null mouse embryonic fibroblasts (PANX1 KO MEFs) transfected with 5 μg human full-length PANX1 or 5 μg human full-length PANX1 and 2.5 μg human PANX11–89; n = 7. (e) Extracellular ATP release from PANX1 KO MEFs transfected with 5 μg human PANX11–89 or 5 μg vector control; n = 8. (f) PANX1-mediated extracellular ATP release from BT549 breast cancer cells expressing PANX11–89 or control vector; n = 8. (g) PANX1-mediated extracellular ATP release from MDA-MB-468 breast cancer cells expressing PANX11–89 or control vector; n = 8. (h) Quantitative RT-PCR analysis of wild-type PANX1 transcript expression in HCC1806 cells expressing PANX11–89 transfected with two independent siRNAs that specifically target full-length PANX1; n = 4. (i) CN34 and CN-LM1A total PANX1 mRNA expression quantified by RNA-seq; FPKM values averaged over two rounds (4 technical replicates) of RNA-seq for each cell line; n = 2. (j) Time-course measurements of ATP release from MDA-MB-231 parental cells and the MDA-LM2 metastatic derivatives sub-lines pretreated with Cbx (500 μM) or PBS for 10 min; n = 4. (k) Quantitative bioluminescence imaging of lung metastasis after the injection of 4 × 104 MDA-LM2 breast cancer cells pretreated with 100 μM 10Panx1 or scrambled peptide, into NOD scid (NS) mice; n = 5. (l) Day 42 quantification of metastatic foci from H&E-stained lungs (left) and representative lung images from vimentin-stained lungs (right) of mice injected with MDA-LM2 cells pretreated with 10Panx1 or scrambled peptide; n = 5. Scale bar, 1 mm. Error bars, s.e.m., ns, nonsignificant, , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents biological replicates ch,l,j, number of mice in k and number of lungs in l. Experiments cg and il were replicated twice with two independent cell lines. Experiments h and i are single experiments with biological replicates. Bioluminescent and histological images are representative of the median.

  3. Supplementary Figure 3: PANX1 activity promotes the metastatic dissemination of breast cancer cells by enhancing early survival in the target organ. (180 KB)

    (a) Daily quantitative imaging plot of lung bioluminescence subsequent to the injection of 4 × 104 metastatic MDA-LM2 breast cancer cells pretreated (30 min) with 100 μM 10Panx1 or scrambled peptides, into NS mice; n = 7. (b) Lungs from mice were extracted at day 3, sectioned and stained for vimentin and the numbers of vimentin-positive cancer cells were quantified; n = 7. Scale bar, 0.25 mm. (c) Quantification of proliferation over 5 days for CN-LM1A and MDA-LM2 cells over-expressing the autoinhibitory C-terminal domain of PANX1 or control vector; n = 4. (d) Quantification of 24 h cell survival for CN-LM1A and MDA-LM2 cells pretreated for 15 min with 100 μM 10Panx1 or scrambled peptide; n = 8. (e) Quantification of 24 h invasion for CN-LM1A and MDA-LM2 cells in the presence of 100 μM 10Panx1 or scrambled peptide; n = 4. (f) Quantification of 36 h anchorage-independent cancer cell survival for CN-LM1A cells in the presence of 100 μM 10Panx1 or scrambled peptide; n = 4. (g) Quantification of 24 h trans-endothelial migration for MDA-LM2 cells in the presence of 100 μM 10Panx1 or scrambled peptide; n = 4. (h) In vivo quantification of luciferase-based caspase-3/7 activity at 3 and 6 h after tail-vein injection of 4 × 104 MDA-LM2 breast cancer cells, pretreated with 100 μM 10Panx1 or scrambled peptide, into NS mice; n = 5. Error bars, s.e.m., , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents number of mice in a,h, number of mouse lungs in b, and biological replicates in cg. Experiments ae and h were replicated with at least two times in two independent cell lines. Experiments f and g are single experiments with biological replicates. Bioluminescent and histological images are representative of the median.

  4. Supplementary Figure 4: PANX11–89 augments ATP release through an interaction with PANX1 to promote breast cancer cell survival in the pulmonary vasculature. (176 KB)

    (a) Quantification of viable, trypan blue-negative CN-LM1A cells with 30 min Boyden chamber centrifugation (3,800 r.p.m.) after cells were pre-treated for 10 min with 500 μM CBX or PBS; n = 4. (b) Quantification of extracellular ATP release from CN-LM1A cells expressing the extracellular ATP hydrolase CD39 or control vector; n = 8. (c) Daily quantitative imaging of lung bioluminescence for three days subsequent to the injection of 1 × 105 CN-LM1A breast cancer cells expressing CD39 or control vector, into NS mice; n = 6. (d) Co-immunoprecipiation of Flag-tagged full-length PANX11−426 and endogenous PANX11–89 from CN-LM1A and MDA-LM2 cells. Anti-PANX1 N-terminal antibody detected a band similar in size to that of Flag-tagged PANX11–89 expressed in HEK293T cells. The presence of this band in the metastatic sub-lines suggests that endogenous PANX11–89 associates with Flag-tagged full-length PANX11−426. The multiple bands representing full-length PANX1 represent the previously described glycosylated forms of PANX1. (e) Co-immunoprecipitation of PANX11–89-Flag from HEK293T cells co-transfected with full-length PANX11−426. Anti-PANX1 N-terminal antibody was used to detect the associated PANX1 species. The multiple full-length PANX1 bands represent the previously described glycosylated forms of PANX1. The input lysates were immunoblotted for PANX1. (f) Increasing concentrations of DSP (dithiobis[succinimidyl propionate]) crosslinker were applied to HEK293T cells expressing Flag-tagged PANX1 before lysis. PANX1 complexes were detected using anti-FLAG M2 antibody. (g) Anti-PANX1 and anti-RFP immunoblotting of DSP crosslinked lysates from HEK293T cells expressing PANX1-EGFP, PANX1-EGFP and PANX11–89-mRFP or PANX11–89-mRFP. Molecular weights are indicated. (h) Quantification of PANX1-mediated ATP release from HEK293T cells transfected with 5 μg control vector, 5 μg wild-type PANX1 (DVVD), 2.5 μg wild-type PANX1 and 2.5 μg PANX11–89, 5 μg caspase resistant full-length PANX1 (AVVA), or 2.5 μg caspase resistant full-length PANX1 and 2.5 μg PANX11–89; n = 4. Error bars, s.e.m., , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents biological replicates in a,b,h, and number of mice in c. Experiments ad were replicated at least two times with at least two independent cell lines. Experiments eh are single experiments with biological replicates. Bioluminescent and images are representative of the median.

  5. Supplementary Figure 5: PANX1 is a druggable target promoting breast cancer metastasis on cancer cell entry into the lung. (370 KB)

    (a) Quantitative imaging of lung bioluminescence 18 h post tail-vein injection of 1 × 106 MDA-MB-468 breast cancer cells, expressing PANX11–89 or a control vector, into NSG mice; n = 6. (b) In vivo quantification of luciferase-based caspase-3/7 activity at 3 and 6 h post tail-vein injection of 1 × 106 MDA-MB-468 breast cancer cells, expressing either PANX11–89 or a control vector, into NS mice; n = 6. (c) Quantification of viable, trypan blue-negative MDA-MDA-468 cells expressing PANX11–89 or a control vector after 1 h extreme hypotonic (12.5% PBS) stretch in the presence of succinate buffer or apyrase (2 U ml−1); n = 4. (d) Quantitative bioluminescence imaging of lung metastasis after tail-vein injection of 1 × 105 CN-LM1A breast cancer cells pretreated for 30 min with CBX (500 μM) or PBS vehicle into NS mice; n = 4 (vehicle), n = 6 (Cbx). (e) Day 35 quantification of metastatic foci (left) and representative lung images (right) from H&E stained lungs of mice injected with CN-LM1A cells pretreated with CBX or PBS vehicle; n = 4 (vehicle), n = 6 (Cbx). Scale bar, 0.5 mm. (f) Mouse body weight before and after daily i.p. injections of CBX (25 mg kg−1) or an equivalent volume of PBS vehicle for seven days. n = 10. (g) 4-week representative images of vimentin-stained lungs from mice treated with two 25 mg kg−1 doses of intravenous CBX or vehicle control. Arrowheads indicate vimentin-positive metastatic foci. Scale bar, 0.5 mm. Error bars, s.e.m., ns, nonsignificant; , P < 0.05; , P < 0.01; , P < 0.001 by a one-tailed Students t-test. n represents number of mice in a,b,d,f, number of mouse lungs in e and biological replicates in c. Experiments ac were replicated at least two times with at least two independent cell lines. Therapeutic PANX1 inhibition experiments were performed using differing protocols with two independent triple negative breast cancer cell lines. Bioluminescent and histological images are representative of the median.

  6. Supplementary Figure 6: Unprocessed scans of immunoblots. (642 KB)

Supplementary Tables

  1. Supplementary Table 1: RNA-seq profile of CN34, CN-LM1A, MDA-MB-231 and MDA-LM2 cell populations. (230 KB)
  2. Supplementary Table 2: Primers used in this study. (419 KB)

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