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. 2013 Feb 1;73(3):1156-67.
doi: 10.1158/0008-5472.CAN-12-1882.

Oncogenic activation of Pak1-dependent pathway of macropinocytosis determines BCG entry into bladder cancer cells

Gil Redelman-Sidi  1 , Gopa Iyer, David B Solit, Michael S Glickman
Affiliations

Oncogenic activation of Pak1-dependent pathway of macropinocytosis determines BCG entry into bladder cancer cells

Gil Redelman-Sidi et al. Cancer Res. .

Abstract

Bacille Calmette-Guerin (BCG) is an attenuated strain of Mycobacterium bovis that is used widely as a vaccine for tuberculosis and is used as an effective treatment for superficial bladder carcinoma. Despite being the most successful cancer biotherapy, its mechanism of action and response determinants remain obscure. Here, we establish a model system to analyze BCG interaction with bladder cancer cells, using it to show that these cells vary dramatically in their susceptibility to BCG infection. Unexpectedly, the uptake of BCG by bladder cancer cells occurs by macropinocytosis rather than phagocytosis. BCG entry into bladder cancer cells relied upon Rac1, Cdc42, and their effector kinase Pak1. The difference in susceptibility between BCG-permissive and -resistant bladder cancer cells was due to oncogenic activation of signaling pathways that activate macropinocytosis, with phosphoinositide 3-kinase inhibitor activation stimulating BCG uptake independently of Akt. Similarly, activated Ras strongly activated Pak1-dependent uptake of BCG. These results reveal that oncogenic activation of macropinocytosis determines BCG uptake by bladder cancer cells, implying that tumor responsiveness to BCG may be governed by the specific mutations present in the treated cancer cell.

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Figures

Figure 1
Figure 1. Heterogeneous BCG susceptibility among bladder cancer lines
A. Specified cell lines were incubated with BCG-GFP for 4 hrs and evaluated by flow-cytometry. For each cell line, representative flow-plots of uninfected and infected cells are shown. X-axis: GFP-fluorescence. Y-axis: pacific-blue fluorescence (empty channel used to facilitate gating). Number within gate represents percentage of GFP-positive events out of total events. B. Specified cell lines were incubated with BCG-GFP for indicated time periods and BCG uptake measured by flow-cytometry. C. Specified cell lines were incubated with BCG-GFP for 24 hours and evaluated by confocal microscopy. Nuclei are stained with Hoechst (blue), actin with Texas-red phalloidin (red) and GFP-expressing BCG is shown in green. Top: 20X magnification. Scale bar: 50 μm. Bottom: 63X magnification. Scale bar: 15 μm. D. Specified cell lines were incubated with BCG-GFP for 4 or 24 hrs, stained with pacific-blue labelled annexin V and evaluated by flow-cytometry.
Figure 2
Figure 2. BCG uptake requires actin and protein kinases
Specified cell lines were pretreated with the indicated small molecule inhibitors and incubated with BCG-GFP for 4 hours in the presence of the inhibitors. BCG uptake was measured by flow-cytometry. BCG uptake is shown as percent of BCG uptake in presence of DMSO. DMSO: Dimethylsulfoxide. CYTO: Cytochalasin D. EIPA: 5-(N-Ethyl-N-isopropyl) amiloride. GENI: Genistein. STAU: Staurosporine. BLEB: Blebbistatin *, P<0.05; **, P<0.005; ***, P<0.0005
Figure 3
Figure 3. BCG uptake requires CDC42, Rac1, and Pak1
A. Specified cell lines were pretreated with IPA-3 or Y-27632 and incubated with BCG-GFP for 4 hours in the presence of the inhibitors. BCG uptake was measured by flow-cytometry. BCG uptake is shown as percent of BCG uptake in presence of DMSO. B. Specified cell lines were stably transduced with empty vector or with DN-Rac1(T17N) or DN-Cdc42(T17N) (both with N-terminal myc-tag). BCG uptake was measured by flow-cytometry 4 hours after infection. Western blot shows expression of myc-tagged Rac1(T17N) or Cdc42(T17N). C. Specified cell lines were stably transduced with empty vector, Rac1(Q61L) or Cdc42(Q61L) (both with N-terminal myc-tag). BCG uptake was measured by flow-cytometry 8 hours after infection. Expression of myc-tagged Rac1(Q61L) or Cdc42(Q61L) was demonstrated by Western blotting. *, P<0.05; **, P<0.005; ***, P<0.0005
Figure 4
Figure 4. BCG uptake by Bladder Cancer Cells requires activated Pak1
A. UM-UC-3 was stably transduced with non-_targeting or two Pak1 shRNAs. BCG uptake was measured by flow-cytometry 4 hours after infection. Knock-down of Pak1 was demonstrated by Western blotting. B. UM-UC-3 was stably transduced with non-_targeting or two Pak2 shRNA. BCG uptake was measured by flow-cytometry 4 hours after infection. Knock-down of Pak2 and lack of significant effect on Pak1 expression was demonstrated by Western blotting. C. Specified cell lines were stably transduced with an empty vector, wild-type Pak1, or DN-Pak1(K299R) (N-terminal myc-tagged). BCG uptake was measured by flow-cytometry 4 hours after infection. Expression of myc-tagged Pak1 was demonstrated by Western blotting. D. Specified cell lines were stably transduced with an empty vector, wild-type Pak1, Pak1(T423E) or Pak1 (L107F) (N-terminal myc-tagged). BCG uptake was measured by flow-cytometry 8 hours after infection. Expression of myc-tagged Pak1 was demonstrated by Western blotting. *, P<0.05; **, P<0.005; ***, P<0.0005
Figure 5
Figure 5. Internalized BCG colocalizes with fluid phase markers
Confocal microscopy of T24 and UM-UC-3 incubated with BCG-GFP for 4 hours in the presence of red-fluorescent dextran (MW 10,000) in the media. Arrows point to location of BCG. Scale bar: 15 μm.
Figure 6
Figure 6. Activation of PI3K stimulates BCG uptake through Pak1 dependent macropinocytosis
A. Western blot of extracts from specified cell lines showing expression of the indicated proteins. B. Specified cell lines were pretreated with wortmannin, Akti XIII or rapamycin and incubated with BCG-GFP for 4 hours in the presence of the inhibitors. BCG uptake is shown as percent of BCG uptake in presence of DMSO. Western blotting of UM-UC-3 after 1 hour treatment with DMSO, wortmannin, Akti XIII, or rapamycin. C. Specified cell lines were stably transduced with non-_targeting or one of two PDK1 shRNAs. BCG uptake was measured by flow-cytometry 4 hours after infection. PDK1 Knock-down was confirmed by Western blotting. D. Specified cell lines were stably transduced with empty vector, PTEN(C124S) or wild-type PTEN. BCG uptake was measured by flow-cytometry 24 hours after infection. Expression of PTEN and downstream pathways was demonstrated by Western blotting. E. Specified cell lines were transduced with non-_targeting or one of two PTEN shRNAs. BCG uptake was measured by flow-cytometry 24 hours after infection. Knock-down of PTEN and its effect on downstream pathways was demonstrated by Western blotting. F. MGH-U4 stably transduced with PTEN shRNA was pretreated with DMSO or IPA-3 and incubated with BCG-GFP for 4 hours in the presence of the inhibitor. BCG uptake was measured by flow-cytometry. *, P<0.05; **, P<0.005; ***, P<0.0005; DMSO: Dimethylsulfoxide. WORT: Wortmannin. AKTI: Akti XIII. RAPA: Rapamycin
Figure 7
Figure 7. Activated Ras stimulates BCG uptake via macropinocytosis
A. Specified cell lines were stably transduced with empty vector, K-ras(G12D) or H-ras (G12V). BCG uptake was measured by flow-cytometry 4 hours after infection. Effect of activated Ras on downstream pathways was demonstrated by Western blotting. B. Phase-contrast and fluorescence microscopy of VMCUB-3 transduced with empty vector, K-Ras(G12D) or H-Ras(G12V), and infected with BCG-GFP for 24 hours. Scale bar: 25 μM. C. Confocal microscopy of VMCUB-3 transduced with empty vector or K-Ras(G12D). Cells were incubated with BCG-GFP for 3 hours in the presence of red-fluorescent dextran (MW 10,000) in the media. Arrows point to location of BCG. Scale bar: 15 μM. D. VMCUB-3 transduced with H-Ras(G12V) was pretreated with DMSO, IPA-3 or wortmannin and incubated with BCG-GFP for 4 hours in the presence of the inhibitor. BCG uptake was measured by flow-cytometry. *, P<0.05; **, P<0.005; ***, P<0.0005; DMSO: Dimethylsulfoxide. WORT: Wortmannin
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