Chloroquine-Inducible Par-4 Secretion Is Essential for Tumor Cell Apoptosis and Inhibition of Metastasis

SUMMARY The induction of tumor suppressor proteins capable of cancer cell apoptosis represents an attractive option for the re-purposing of existing drugs. We report that the anti-malarial drug, chloroquine (CQ), is a robust inducer of Par-4 secretion from normal cells in mice and cancer patients in a clinical trial. CQ-inducible Par-4 secretion triggers paracrine apoptosis of cancer cells and also inhibits metastatic tumor growth. CQ induces Par-4 secretion via the classical secretory pathway that requires the activation of p53. Mechanistically, p53 directly induces Rab8b, a GTPase essential for vesicle transport of Par-4 to the plasma membrane prior to secretion. Our findings indicate that CQ induces p53- and Rab8b-dependent Par-4 secretion from normal cells for Par-4-dependent inhibition of metastatic tumor growth.

Figure S1. CQ induces Par-4 secretion from normal cells, but not cancer cells, by an apoptosis-independent mechanism, Related to Figure 1.
A. Screen for Par-4 secretagogues. MEFs were treated with 25 M amounts of generic compounds or vehicle (V) control for 24 h. The CM and whole-cell lysates were subjected to Western blot analysis with the indicated antibodies. Collagen1A1 (Col1A1) was used as a loading control for protein secretion, as it was generally unchanged in response to the treatments. The samples were also subjected to SDS-PAGE and Coomassie blue staining for albumin levels in serum from the CM, which was another loading control. Actin served as a loading control for the lysates.
B. CQ induced Par-4 secretion by an apoptosis-independent mechanism and Par-4 did not regulate expression of the autophagy associated protein p62/SQSTM1. HEL cells were pre-treated with zVAD-fmk (2 M) or Vehicle (V) for 30 minutes, and in the presence of zVAD-fmk, further treated with CQ (20 M) or V or directly treated for 24 h with V or CQ in the absence of any pretreatment. The CM and lysates were analyzed on Western blots (a). MEF cells or HEL cells were treated with CQ (25 M) or V for 24 h. Percent viability in the cultures was determined by MTS Cell Proliferation Colorimetric Assay (b) and percent apoptosis was determined by ICC for active caspase 3 and DAPI staining (c). Wild type MEFs (p53 +/+ ) were treated with CQ (25 M) or vehicle for 24 h, stained with propidium iodide, and cell cycle distribution was analyzed in the Becton-Dickinson LSRII flow cytometer at the Markey Cancer Center Flow Cytometry shared resource facility (d). Par-4 +/+ and Par-4 -/-MEFs were treated with vehicle or CQ (20 M) for 24 h, and whole cell extracts were examined for p62 expression by Western blot analysis (e).

B. Plasma from CQ treated patients induces ex vivo apoptosis of cancer cells.
Aliquots of pre-CQ or post-CQ treatment plasma samples (20% final concentration) from patient RCC4 were transferred to cancer cell cultures or normal cells. Apoptotic cells were scored after 24 h. FBS, fetal bovine serum, was used as another control. * P<0.0001 by the Student's t-test.

A. Par-4 in CM from CQ treated MEFs induced apoptosis in LLC1 cells.
Aliquots of CM from Par-4 +/+ or Par-4 -/-MEFs treated for 24 h with CQ (20 M) or vehicle (v) were incubated with LLC1 cells (left panel). Moreover, aliquots of CM from wild type MEFs treated with CQ or vehicle were incubated with control (C) antibody (Ab), Par-4 (P) Ab or GRP78 (G) Ab and then transferred to LLC1 cells (right panel). After 24 h, the cells were scored for apoptosis (left and right panels). * P<0.0001 by the Student's t-test.

b. CQ induced Par-4 secretion is essential for inhibition of metastasis by CQ in
the EO771 experimental metastasis model. Athymic (nu/nu) mice were injected i.v. with EO771 cells (0.5 x 10 6 cells), and 24 h later injected i.p. with CQ (25 mg/kg body weight) or vehicle (V) once every day for 5 consecutive days. Animals injected with CQ were also injected with either the control IgG or Par-4 polyclonal antibody (20 g/injection). After 21 days, the lungs were perfused, stained with India ink (upper panel) and the tumor nodules were scored (lower panel). * P < 0.001 by the Student's ttest.
c. CQ did not inhibit the growth of subcutaneous bulky tumors. We used 4 x10 5 H460 or LLC1 cells to generate subcutaneous tumors in the flanks of immunocompromised (nu/nu) mice. When the tumors had grown to about 200 mm 3 volume (indicated by arrow), the mice were injected with CQ (25 mg/kg body weight) or vehicle once daily throughout the experiment (10 mice per group). Tumor growth was followed over period of the experiment, and average tumor volumes + SD in each cohort are shown. The difference in tumor volume between the vehicle and CQ treated groups was not significant (n.s., P > 0.05) by the Student's t test. A. CQ induced p53-dependent transcription and upregulation of p53-responsive genes. MEFs (p53 +/+ or p53 -/-) were transiently co-transfected with p53-reporter (PG13luc containing p53 binding sites), mutant reporter (MG15-luc containing mutated binding sites for p53) or pGL3 control luc construct, and -galactosidase expression construct. The transfectants were treated with CQ (25 M) or vehicle for 24 h, luciferase activity was determined and normalized to corresponding -galactosidase activity and expressed as relative luciferase activity units. CQ induced luciferase activity from the p53-reporter luciferase construct but not from a luciferase construct containing mutant response element in p53 +/+ MEFs. By contrast, CQ failed to induce luciferase activity in p53 -/-MEFs.
B. CQ induced upregulation of p53-responsive genes. C57BL/6 mice were injected once daily with CQ (25 mg/kg body weight) or vehicle for five consecutive days. Blood samples were collected from the mice on the sixth day, and plasma was examined for expression of p53, p21, PIG3, Par-4, and actin by Western blot analysis.

C. CQ did not elevate Par-4 RNA levels.
MEFs were treated with CQ (25 M) or vehicle (V) for 24 h and mRNA prepared from the cells was examined by Real-Time quantitative reverse transcription PCR (qRT-PCR) for Par-4. The data were normalized relative to a GAPDH control.

D. CQ inhibited NF-B activity, but not AP1 activity in normal cells.
Normal (HEL and MEF) or cancer (LLC1, H460, A549, PC-3) cells were co-transfected with luciferase (luc) reporter construct for NF-B, AP1, or empty luc construct and -galactosidase expression construct and treated for 18 h with CQ (25 M) or vehicle (V). Luciferase activity was analyzed in the cell lysates and normalized with respect to the corresponding -galactosidase activity.
A and D. * P < 0.001 by the Student's t-test.    Figure 6C, were treated with vehicle (V) or CQ (25 M) in the absence or presence of BFA (1 g/ml) for 24 h, and subjected to ICC for Par-4 (red fluorescence) and Rab8b (green fluorescence). Cells were stained with DAPI to reveal their nuclei (cyan fluorescence). The percentage of cells showing colocalization of Par-4 and Rab8b vesicles was quantified (lower right panel). In HEL cells, note dissociation of Par-4 and Rab8b (loss of yellow fluorescence, but retention of red and green fluorescence) in the CQ + BFA panel. As expected, Rab8b -/-MEF cells showed absence of Rab8b staining with the Rab8b antibody. * P < 0.001 by the Student's t-test.

Chromatin-Immunoprecipitation (ChIP) Assay
ChIP assays for CQ-induced or vehicle-treated basal levels of p53 binding to the Rab8b promoter or to Par-4 gene involved treatment of wild type MEFs (2 x10 6 cells) with vehicle or CQ (25 uM) for 8-12 Hrs. ChIP analysis was performed by using the ChIP Assay kit from Millipore or Active Motif according to the instructions provided by the manufacturer. Sheared chromatin was immunoprecipitated with antibodies using mouse IgG (sc-2025, Santa Cruz Biotechnology), or anti-p53 antibody (1C12 mouse monoclonal antibody #2524 from Cell Signaling. DNA fragments were amplified by using validated primers for p53 binding site on the Rab8b promoter from EpiTect ChIP qPCR primer assay for mouse Rab8b (GPM1054933(-)01A; Qiagen) and resolved on agarose gels, or quantitative PCR was performed on CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). Two different primer sets: (1) Forward 5' TCACCTGAGCAAAACGACGA 3'; Reverse 5' GCGATCAGAGCTAAGGGGAC 3'; (2) Forward 5' GGCTGAGCCTGTCCTCTTTC 3'; Reverse 5' GTGCTGTGTTGGTTTCTCGG 3'; were used along the Par-4 gene, which does not contain a p53-binding site. GAPDH control primers used were: Forward 5'-ATG GTT GCC ACT GGG GAT CT-3'; Reverse 5'-TGC CAA AGC CTA GGG GAA GA-3'.

Animal experiments
To determine whether CQ induced Par-4 secretion in immunocompetent mice, C57BL/6 mice were injected via the intraperitoneal (i.p.) route with a single injection of CQ (50 mg/kg body weight) or vehicle, and whole-blood samples were collected 24 h later. Plasma was separated from the blood samples, heated at 56 o C to inactivate complement. Aliquots of the mouse plasma samples were added to the growth medium (final 20% mouse plasma) of normal and cancer cells in culture and tested for induction of ex vivo apoptosis in cancer cells. To test the effect of CQ on metastatic growth of tumors, EO771 cells or LLC1 cells expressing luciferase (0.5 x 10 6 cells) were injected via the tail vein in Par-4 +/+ (wild type) or Par-4 -/-C57BL/6 mice and 24 h later, the mice were injected i.p. with CQ (25 mg/kg body weight) injection given daily for five consecutive days. Each group included 8 mice. Plasma from the mice was examined 24 h after the 5th CQ injection for Par-4 expression. The mice were imaged for luciferase expression as previously described (Zhao et al., 2011), and humanely killed to examine their tumors at day 21. The lungs were then perfused, stained with India ink and the tumor nodules were scored as previously described (Zhao et al., 2011).
To test whether Par-4 secretion induced by CQ was involved in inhibition of LLC1-or EO771-derived metastatic tumor growth, we used an experimental metastasis model. Athymic (nu/nu) mice were injected i.v. with 500 x 10 5 LLC1 cells, and 24 h later injected i.p. with CQ (25 mg/kg body weight) or vehicle once every day for 5 consecutive days. Animals injected with CQ were also injected within 2 h with either the control IgG or Par-4 polyclonal antibody (20 g/injection). Each group included 10 mice. After 21 days, the lungs were perfused, stained with India ink and the tumor nodules were scored.
To determine whether CQ inhibits the growth of large bulky tumors, we injected 4 x 10 5 H460 or LLC1 cells subcutaneously in nude (nu/nu) mice to generate tumors in the flanks. When the tumors had grown to ca. 200 mm 3 volume (indicated by arrow), the mice (10 per group) were injected with CQ (25 mg/kg body weight) or vehicle once daily throughout the experiment, and tumor growth was measured with calipers over period of the experiment to calculate tumor volume. All animal procedures were performed with University of Kentucky IACUC approval.