WO2019143934A1 - Anti-cancer activity of scbg3a2 and lps - Google Patents

Abstract

Methods of treating cancer with a combination of SCGB3A2 and LPS are described. The cancer can be, for example, a SDCl positive cancer and/or a cancer that expresses genes and proteins of the non-canonical inflammasome. Pharmaceutical compositions that include SCGB3A2 and LPS are also described.

Description

ANTI-CANCER ACTIVITY OF SCBG3A2 AND LPS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claim the benefit of U.S. Provisional Application No. 62/619,511, filed January 19, 2018, which is herein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to use of secretoglobin 3A2 (SCGB3A2) and

lipopolysaccharide (LPS) for the treatment of cancer, for example for the treatment of lung or colon cancer.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government Support under project number Z01BC010449 awarded by the National Institutes of Health, National Cancer Institute. The Government has certain rights in this invention.

BACKGROUND

SCGB3A2, also called uteroglobin-related protein 1, is a member of the secretoglobin (SCGB) gene superfamily, and has been identified as having growth factor, anti-inflammatory and anti-fibrotic activities. All members of the SCGB gene superfamily are cytokine-like secreted proteins of approximately 10 kDa, found only in mammals. SCGB3A2 is the second member of the SCGB family 3, subfamily A, and is predominantly expressed in lung airways. Prior studies using mouse models showed that SCGB3A2 suppresses lung inflammation and promotes branching and maturation of mouse fetal lungs.

Lipopolysaccharide (LPS) is a large molecule comprising a lipid and a polysaccharide found in the outer membrane of Gram-negative bacteria. The presence of LPS in mammalian tissues is known to elicit an immune response, and in some instances, induce pyroptosis through the non-canonical inflammasome.

SUMMARY OF THE DISCLOSURE

This disclosure provides methods of using SCGB3A2 protein in combination with LPS for treating cancer in a subject. The utility of the combination of LPS and SCGB3A2 for treatment of cancer was previously unknown, and is surprising as SCGB3A2 was previously identified as an anti-inflammatory molecule, but when combined with LPS facilitates“inflammation-induced death” of cancer cells.

In some embodiments, a method is provided for treating or preventing cancer in a subject. The method comprises administering a therapeutically effective amount of LPS and a SCGB3A2 protein to a subject with cancer or at risk of cancer to inhibit the cancer in the subject. In some embodiments, the cancer is a syndecan-l (SDC1) positive cancer and/or is a cancer that expresses genes and proteins involved in a non-canonical inflammasome (for example, the cells of the cancer express CASP4 and/or CASP5 ). In several embodiments, the cancer is a lung cancer or a colorectal cancer.

Additionally, this disclosure provides methods of using SCGB3A2 protein for treating cancer in a subject, wherein the cancer is not lung cancer. The method comprises administering a therapeutically effective amount of SCGB3A2 protein without LPS to a subject with cancer or at risk of cancer to inhibit the cancer in the subject, wherein the cancer is not lung cancer. In some embodiments, the cancer is a syndecan-l (SDC1) positive cancer and/or is a cancer that expresses genes/proteins of a non-canonical inflammasome (for example, the cells of the cancer express CASP4 and/or CASP5 ). In several embodiments, the cancer is a colorectal cancer.

Pharmaceutical compositions comprising SCGB3A2 and LPS are also provided. In some embodiments, the pharmaceutical composition is formulated for oral administration and delayed release in the intestine. In other embodiments, the pharmaceutical composition is formulated for administration by inhalation.

The foregoing and other features and advantages of this disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1I. SCGB3A2-induced suppression of Lewis lung carcinoma (LLC) cell proliferation. (FIG. 1A) Effect of SCGB3A2 on proliferation of LLC cells. Cells were maintained without serum for 24 hours, followed by 1% FBS-RPMI1640 media with or without mouse SCGB3A2 (1 pg/ml). Cell Counting Kit 8 (CCK8) assay was carried out 72 hours after the addition of SCGB3A2. Averages ± SD from three independent experiments. **: P<0.0l by student’s t-test. (FIG. 1B) LLC cell intravenous metastasis model scheme. Mice inoculated with LLC cells received daily intravenous administration of mouse recombinant SCGB3A2 for 7 consecutive days for the I^st, 2^nd, or 3^rd week, or the entire experimental period of 21 days. Control mice received PBS alone. The number of the pulmonary surface tumors was counted on day 21. (FIG. 1C) Representative lung images from each SCGB3A2 administration group. Scale bar = 1 cm. (FIG. 1D) Number of pulmonary surface tumors. Each dot represents one mouse. *: P<0.05, **:P<0.0l. (FIG. 1E) Number of pulmonary surface tumors larger than 3 mm. *: P<0.05, **:P<0.0l. (FIG. 1F) (Upper panel) Representative pictures of metastasized lung tumors from wild-type (WT) and Scgb3a2- null ( Scgb3a2( -/-)) mice. Scale bar = 1 cm. (Lower panel) H&E staining of lungs. Scale bar = 500 pm. (FIG. 1G) Representative lungs from WT littermate, Scgb3a2- null ( Scgb3a2( -/-)), and Scgb3a2- null mice given SCGB3A2 (Scgb3a2(-/-)+ SCGB3A2) for the I^st week. This was a separate independent experiment from those presented in FIGS. 1C- 1E. WT and Scgb3a2(-/-) mice received daily PBS as a control. Lung necropsy was carried out on day 21. (FIG. 1H) Graph showing the number of pulmonary surface tumors of experiment in FIG. 1G. (FIG. II) Lung weight of each LLC cell metastasis model in FIG. 1G. KO: Scgb3a2 -null; *: P<0.05. Statistical differences calculated by One-way ANOVA except in FIG. 1A.

FIGS. 2A-2G. SCGB3A2 as an LPS binding protein. (FIG. 2A) CCK8 analysis using various concentrations as shown (pg, ng/ml) of smooth LPS ( E . coli 011 LB4 serotype) and rough A LPS (Ra-LPS) after 72 hours in culture. C: control without any addition of LPS. (FIG. 2B) Reverse staining of aggregation of LPS. Imidazole-zinc staining of E. coli 011 LB4 serotype LPS on agarose gel. LPS (10 pg) was incubated with human SCGB3A2 in lane 1 to 5: 0 ng, 10 ng, 100 ng, 1 pg and 10 pg, respectively. Arrows indicate the bottom of the aggregate or smeary bands. (FIG. 2C) Reverse staining of aggregation of LPS. Imidazole-zinc staining of E. coli 011LB4 serotype LPS on agarose gel. Bovine serum albumin (BSA) 10 pg (lane 1), human SCGB3A2 10 pg (lane 2), LPS 10 pg (lane 3), BSA+LPS pre-incubation at 37°C, 30 minutes (lane 4),

SCGB3A2+LPS pre-incubation at 37°C, 30 minutes (lane 5), SCGB3A2+LPS pre-incubation at room temperature (RT), 30 minutes (lane 6), SCGB3A2+LPS pre-incubation at 37°C, 10 minutes (lane 7), SCGB3A2+LPS pre-incubation at RT, 10 minutes (lane 8). Bottom image is Coomassie Brilliant Blue (CBB) staining of the same gel. (FIG. 2D) Streptavidin pull-down assay of LPS- Biotin and recombinant SCGB3A2. Immunopreeipitation (IP) and western blotting were sequentially carried out using anti-SCGB3A2 and anti-LPS antibody, respectively. Input is 10%. (FIG. 2E) Dynamic light scattering (DLS) assay. Size deformation of LPS micelles by human SCGB3A2 pre-incubation. Histogram shows the intensity of hydrodynamic radii (nm) of 0111:B4 LPS (20 pg/ml), human SCGB3A2 (20 pg/ml), and LPS pre-incubated with SCGB3A2 for 30 minutes at RT. Gel analysis and DLS assay were carried out more than three separate times and each time, similar results were obtained. (FIG. 2F) Effect of SCGB3A2 or LPS on the number of lung surface tumors in LLC cell intravenous metastasis model. LPS(C3): LPS concentration equivalent to that contained in mouse SCGB3A2(C3); SCGB3A2(C1): human SCGB3A2(C1) protein without addition of exogenous LPS (Cl and C3; see Table 1); LPS(Ci): LPS concentration equivalent to that contained in human SCGB3A2(C1); and LPS high: LPS (1 qg/mouse). Each dot represents one mouse. Averages ± SI) are shown. **p<0.01. (FIG. 2G) Representative images of lung of mice with SCGB3A2(C3) or LPS(C3) administration. Bar = 300 pm.

FIGS. 3A-3H. SCGB3A2 binding to LLC cells through heparan sulfate (HS) chains of syndecan-1 (SDC1). (FIG. 3A) Schematic model of a human protein array for the isolation process of candidate genes shown as a Venn diagram. (FIG. 3B) Co-IP assay of SCGB3A2-FLAG and SDCl-Myc-His in COS-1 cells. IP and western blotting were sequentially carried out using anti- FLAG and anti-Myc antibody, respectively. (FIG. 3C) SCGB3A2 and SDC1 immunostaining in the airway epithelial cells of adult wild-type mouse lungs. Counterstained with hematoxylin. Bar = 10 qm. (FIG. 3D) Immunofluorescent staining of SDC1 in LLC and B16F10 cells grown in 10% FBS-RPMI 1640 medium for 24 hours. DAPI was used for nuclear staining. Arrow indicates SDC1 cell surface expression in LLC. Arrowheads point to staining at the cell-cell junction or focal SDC1 staining near the nucleus (as indicated) in B16F10 cells. Bar = 20 qm. (FIG. 3E) Flow cytometric analysis for SDC1 expression on the cell surfaces of LLC and B16F10 cells using phycoerythrin (PE) -conjugated anti-SDCl ectodomain specific antibody. (FIG. 3F) Flow cytometric analysis for SCGB3A2 binding to LLC cells using anti-SCGB3A2 antibody.

Glutathione-S-transferase (GST) tagged mouse SCGB3A2 (3 qg) was incubated with LLC cells at 4°C for 30 minutes, followed by rabbit anti-mouse SCGB3A2 antibody. Cells were stained with PE-anti-rabbit IgG antibody at 4°C for 30 minutes. (FIG. 3G) SCGB3A2 binding assay on LLC- sh-Control or sh-SDCl cells. GST tagged mouse SCGB3A2 (1 qg) was incubated with each cell type at 4°C for 30 minutes, followed by staining with Alexa 488 anti-rabbit IgG antibody at 4°C for 30 minutes. (FIG. 3H) SCGB3A2 binding assay on LLC cells. Cells were co-incubated with or without GST tagged mouse SCGB3A2 (1 qg) or SCGB3A2+heparin. Cells were stained with PE- anti-rabbit IgG antibody at 4°C for 30 minutes.

FIGS. 4A-4P. SCGB3A2-LPS uptake activates inflammasome signaling. (FIG. 4A) Immunofluorescence analysis of LLC and ARH-77-mSDCl cells for SCGB3A2, SDC1, and ICAM-1. Arrowheads: uropod-like structures. Cells were incubated in 0% FBS-RPMI 1640 for 40 minutes (0%40m) with or without GST-mSCGB3A2 (1 qg). Scale bar = 10 qm. (FIG. 4B) Immunofluorescence analysis of LLC cells for clathrin and HT (HaloTag). Cells were incubated with LPS_A488 (1 qg/ml) and SCGB3A2-HT supernatant (SN) in 0%lh. Arrowheads: uropod structure. Bar = 10 qm. (FIG. 4C) SCGB3A2 tetramer model (see also FIGS. 11A-110). Left: Exploded view of the tetramer model showing the two dimers, which are shaded to identify the two monomers. The dimer structure reveals a pocket accessible from Face A and flanked by positively charged residues (Gly 1 -N termini-; Arg 6 and Lys 61; Face A also shown in FIG. 111) forming a pattern consistent with a heparan or LPS binding motif. Face A is exposed in the tetramer while Face B (right) is occluded (Face B also shown in FIG. 11J). (FIG. 4D) Immunofluorescence analysis of LLC cells maintained in 0%5 h for EEA1, LAMP1 and LPS_A594. Arrowheads:

overlapping staining of LPS_A594 and EEA1. Bar = 10 pm. (FIG. 4E) Immunofluorescence analysis of LLC cells after 1 % 16 h incubation with LPS_A594 (2 pg/ml) and SCGB3A2 (2 pg/ml).

Arrowheads: cytosolic LPS signal, not overlapping with EEA1 and LAMP1 staining. LP: LPS, E: EEA1, LA: LAMP1. Bar = 10 pm. (FIG. 4F) Immunofluorescence analysis of LLC cells for LPS_A594, EEA1, SDC1 after 1%16 h incubation with LPS_A594 (2 pg/ml) and SCGB3A2 (2 pg/ml). Arrowheads: cytosolic LPS signal, not overlapping with EEA1 and SDC1 staining. Bar = 10 pm. (FIG. 4G) Immunoblots of LLC-sh-TLR4 cells after treatment with or without LPS (011 LB4, 10 ng/ml) or SCGB3A2 (200 ng/ml) for 3 and 24 hours in OPTI-MEM. (FIG. 4H) Immunoblots of RAW264.7 cells after treatment with or without LPS (011 LB4, 1 pg/ml), SCGB3A2 (1 pg/ml), and/or heparin (1 pg/ml) for 16 hours in OPTI-MEM. Abbreviations for FIG. 2G and FIG. 2H, L: LPS, S: SCGB3A2, H: Heparin. (FIG. 41) Immunofluorescence analysis of LLC cells for caspasel 1 and NLRP3, incubated for 0 %5 h with or without LPS_A594 (2 pg/ml), LPS_A594+

SCGB3A2 (2 pg/ml). Arrowheads: LPS, caspase 11, and NLRP3 overlapping focus. Bar = 10 pm. (FIG. 4J) Immunofluorescence analysis of caspase 11 in LPS -transfected LLC. LPS_A594 (1 pg) was transfected using X-tremeGENE HP in 10%5 h. Arrowheads: LPS and caspase-11 focus. Bar =

10 pm. All images are the representative of three independent experiments. (FIG. 4K)

Immunofluorescence analysis of LLC cells for SCGB3A2-HT and LPS_A488. Cells were incubated for 1%24 hr with or without Dynasore (10 pM), and LPSA4SS (1 pg)+SCGB3A2-HT supernatant. Dotted lines depict the outer membrane of cells. Bar = 10 pm. (FIG. 4L) Immunoblots of LLC cells after treatment with or without LPS (Oil l:B4, 10 ng/ml), SCGB3A2 (1 pg/ml) or Dynasore (5 or 10 pM) for 2%3 hr. (FIG. 4M) LDH release from LLC cells in control condition or in the presence of Dynasore (D, 5 pM) or Wedelolactone (W, 10 pM), with or without LPS (1 pg/ml) and/or SCGB3A2 (1 pg/ml) for 2%48 hr. Average ±SD from three independent experiments, each in triplicate. C: control, S: SCGB3A2, L: LPS, L + S: LPS +SCGB3A2. *: p<0.Q5 by one-way ANQVA (FIG. 4N) Number of pulmonary surface tumors in lungs of intravenous metastasis model mice using LLC-control or LLC-sh-Casp-l 1 cells and indicated treatments. hS2; human SCGB3A2. Each dot represents a single mouse. **: p O.Ol by student’s r-test. (FIG.

40) Immunoblots of LLC cells for caspase-11 and GAPDH after treatment with or without LPS (L, 10 ng/ml), SCGB3A2 (S, 200 ng/ml), Wedelolactone (W, 50 pM) in 2%3 hr. C: control. (FIG. 4P) Immunoblots for caspase-11 and GAPDH using cell lysis of control LLC or sh-caspl l shRNA transfected LLC cells after treatment with or without LPS (L, 10 ng/ml), SCGB3A2 (S, 200 ng/ml) in 2%3 hr. C: control.

FIGS. 5A-5F. SCGB3A2-LPS promotion of pyroptotic cell death. (FIG. 5A) Phase contrast image of LLC cells incubated with LPS (0111 :B4, 1 pg/ml) and SCGB3A2 (1 pg/ml) in 1% FBS-RPMI 1640 for 72 hours. Arrows indicate cells undergoing pyroptosis (swollen cells).

Bar = 10 pm. (FIG. 5B) CCK8 analysis using LLC cells with LPS (Oll l:B4) (1 ng/ml) and/or human SCGB3A2 (C2; see Table 1) (10 ng/ml) in 2% FBS-RPMI for 72 hours culture. Data are the representative from more than three independent experiments. **p<0.0l, *p<0.05 by One-way ANOVA. S2: SCGB3A2. (FIG. 5C) Flow cytometry analysis for Annexin V/PI staining. One pg/ml SCGB3A2 and 10 ng/ml LPS (Ol ll:B4) were used. LLC cells were maintained for 24 hours in 1% FBS-RPMI medium, then treated with or without SCGB3A2/LPS, and further cultured for 48 hours. (FIG. 5D) TUNEL staining of lung sections of lung metastasized LLC cells in the intravenous administration model. Images are shown for control (PBS) and mouse SCGB3A2 administered during the 2nd week. The bottom graph indicates the percentage of TUNEL positive areas per total LLC tumor areas as measured using imageJ. +SCGB3A2 indicates lungs of mice that received SCGB3A2 during the I^st week. Ctl, control. *p<0.05 by student’s t-test. Three independent lung samples were evaluated for each group. (FIG. 5E) Representative IHC staining of caspase-11 for metastasized nodules of LLC cells in lung in vivo. Images are shown for control (PBS) and mouse SCGB3A2 administered during the lst week. Counterstained with hematoxylin. Bar = 50 pm. (FIG. 5F) Representative IHC staining of NLRP3 for metastasized nodules of LLC cells in lung. Images are shown for control (PBS) and mouse SCGB3A2 administered during the lst week. Counterstained with hematoxylin. Bar = 50 pm.

FIGS. 6A-6F. Evaluation for the requirement of SDC1 and other genes for SCGB3A2-LPS effect. (FIG. 6A) CCK8 assay using LLC-sh-Control and LLC-sh-SDCl for 72 hours in 1% FBS- RPMI 1640 medium. C: control, S: human SCGB3A2 (200 ng/ml), L: LPS (Oll l:B4, 1 pg/ml). Data are the representatives of more than three independent experiments. *: P<0.05 by one-way ANOVA. (FIG. 6B) Immunofluorescent staining of caspase-11 and LPS_A594 using LLC-sh-Control and LLC-sh-SDCl cells. Cells were maintained in 1% FBS-RPMI 1640 medium for 16 hours with or without human SCGB3A2 (1 pg/ml), LPS (1 pg/ml), and/or heparin (1 pg/ml). Arrowheads: caspase-11 foci. Bar = 10 pm. Numbers on the right indicate cells containing caspase-11 foci per a total 100 cells counted for each cell type. Data are the representatives of three independent experiments. (FIG. 6C) Intravenous metastasis model using LLC-sh-Control or LLC-sh-SDCl cells. Representative lungs from the control (PBS) and SCGB3A2 groups that received treatment in the lst week. Bar = 1 cm. (FIG. 6D) Summary for the numbers of pulmonary surface tumors in lungs of animals receiving treatment as indicated. hS2: human SCGB3A2, mS2: mouse SCGB3A2. Each dot represents a single mouse. *: P<0.05 by One way ANOVA. (FIG. 6E) qPCR

quantification of the relative expression levels for inflammasome related genes in LLC and B16F10 cells. Cells grown in 10% FBS-RPMI 1640 medium were harvested at 24 hours. Graphs are the representatives of three independent experiments, each in triplicate. ND: not detectable. (FIG. 6F) Kras^G12D - duce lung carcinogenesis survival curve for Kros^GI2D;Scgb3a2(fl/fl) (fl/fl, n = 14) and littermate Kras^G12D ;Scgb3^' a2(jl/+ ): fl/+, n = 44) mice.

FIGS. 7A-7I. Evaluation for the susceptibility to SCGB3A2-LPS and expression analysis of SDC1 and caspase-4 in various human cancer cells. (FIG. 7 A) qPCR quantification of the relative expression levels for SDC1 in various human malignant cells. Cells grown in 10% FBS- RPMI 1640 medium were harvested at 24 hours. The expression level of A549 cell was arbitrarily set as 1.0. Graphs are the representatives of three independent experiments, each in triplicate. (FIG. 7B) qPCR quantification of the relative expression levels for CASP4 in various human malignant cells. Cells grown in 10% FBS-RPMI 1640 medium were harvested at 24 hours. The expression level of A549 cell was arbitrarily set as 1.0. Graphs are the representatives of three independent experiments, each in triplicate. (FIG. 7C) Combined qPCR results of (FIG. 7A) and (FIG. 7B). Number shows the rank of expression from the highest as 1. Graph is based on the order of CASP4 expression. The susceptibility to SCGB3A2+LPS determined by CCK8 assay is shown as“y (observed)” or (not observed)” in CCK8 row. (FIG. 7D) Correlation graph between SDC1 and CASP4 in 20 human cancer cells. Pearson correlation r = 0.7988. P<0.0001. (FIG. 7E)

Representative CCK8 analysis results using 7 human cancer cells. Cells were incubated with SCGB3A2 (1 pg/ml) and/or LPS (0111:B4, 100 pg/ml) for 48 hours. C: control, S2: SCGB3A2, L: LPS. *: P<0.05, **:P<0.01 by One-way ANOVA. (FIG. 7F) Representative immunofluorescence analysis using 7 human cancer cell lines. Counter stained with DAPI. Bar = 10 pm. Arrowheads indicate the membranous SDC1 expressions. (FIG. 7G) Flow cytometric analysis for SDC1 expression on cell surfaces of 7 human cancer cells using anti-SDCl antibody. Solid histograms indicate unstained negative control. (FIG. 7H) Flow cytometric analysis for HS expression on cell surfaces of 7 human cancer cells using anti-SDCl antibody. Solid histograms are unstained negative control. (FIG. 71) Schematic model for LPS entry into cells by SCGB3A2 through SDC1 receptor, leading to pyroptotic cell death.

FIGS. 8A-8E. Analysis of LPS-SCGB3A2 complex. (FIG. 8A) CCK8 analysis using various recombinant SCGB3A2s (1 pg/ml) obtained from different sources/batches. LLC cells grown in 1% FBS-RPMI 1640 medium were harvested at 72 hours and analyzed. Data are the representative from more than three experiments. S2: SCGB3A2. For Cl, C2, and C3, see Table 1. (FIG. 8B) Reverse staining of aggregation of LPS. Imidazole-zinc staining of LPS from E. coli EH 100 (Ra mutant) (land and 2), LPS from Salmonella typhimurium (lane 3 and 4), LPS from E. coli K235 (lane 5 and 6). Each form of LPS (10 pg) was incubated with human SCGB3A2 (10 pg) in lane 2, 4 and 6. Asterisks (*) indicate the size of background staining of loading dye. Left arrow points to the small size band appearing upon addition of SCGB3A2. Center and right arrows indicate the appearance of small size smeary band upon addition of SCGB3A2. (FIG. 8C) DLS analysis using LPS (011 LB4, 20 pg/ml) and pre-incubation with SCGB3A2, 0 as control, 0.2, 2, and 20 pg/ml. (FIG. 8D) DLS analysis using E. coli K235 serotype LPS (20 pg/ml) without and with human SCGB3A2 (20 pg/ml). (FIG. 8E) DLS results for Ra-LPS (20 pg/ml) with or without pre-incubation with human SCGB3A2 (20 pg/ml).

FIGS. 9A-9F. Cell surface expression of SDC1 and validation of sh-SDCl and ARH-77- mSDCl clones. (FIG. 9A) Representative IHC for SDC1 in lung metastasized LLC cells in mice. Bar = 200 pm (upper), 50 pm (bottom). (FIG. 9B) LLC and B16F10 cells grown in 10% FBS- RPMI 1640 for 24 hours were subjected to subcellular fractionation and SDC1 expression on the cell membrane fraction was examined by western blotting using anti-SDCl ectodomain specific antibody. (FIG. 9C) Left: FACS analysis for mouse SDC1 expression using LLC-sh-Control and two different LLC-sh-SDCl clones. Right: qPCR analysis for Sdc 1 , Sdc2 and Sdc4 mRNA expressions using LLCsh-Control and two different sh-SDCl clones. Accordingly, sh-SDCl(A) clone was used in the current studies. (FIG. 9D) Validation of mouse SDC1 expressions on ARH- 77-mSDCl cells using anti-mouse SDC1 antibody. Images are the representatives of two independent experiments. Bar = 10 pm. (FIG. 9E) SCGB3A2 binding assay on ARH-77-control or ARH-77-mSDCl cells. GST tagged mouse SCGB3A2 (1 pg) was incubated with each cell type at 4°C for 30 minutes, followed by staining with PE-anti-rabbit IgG antibody at 4°C for 30 minutes. (FIG. 9F) SCGB3A2 binding assay on ARH-77-mSDCl cells. Cells were co-incubated with or without GST tagged mouse SCGB3A2 (1 pg) or SCGB3A2+heparin.

FIGS. 10A-10D. Analysis of SCGB3A2-HaloTag protein trafficking. (FIG. 10A) Live cell imaging of LPS_A488 binding to LLC cells. LLC cells were incubated in 1% FBS-RPMI1640 with LPS_A488 (1 pg/ml) for 16 hours with HaloTag control vector- transfected supernatant (SN) or mouse SCGB3A2 HaloTag (HT) construct-transfected SN. Arrows indicate the binding of LPS_A488 on cell surfaces. Images are overlaid with phase contrast. Bar = 10 pm. (FIG. 10B) Tmmunoblot for HaloTag protein using cell lysis or culture supernatant of HEK293 cells transfected with HaloTag control vector or mouse SCGB3A2-HT vector. (FIG. 10C) Immunofluorescence staining using Halotag®TMRDirect ligand and anti-clathrin antibodies. LLC cells were incubated with or without SCGB3A2-HT supernatant and TMRDirect ligand in 1% FBS-RPMI 1640 for 16 hours. After fixation, cells were stained with anti-clathrin antibody. Arrowheads indicate polarized clathrin staining. Bar = 10 pm. (FIG. 10D) Live cell imaging using Halotag® TMR Direct ligand. LLC-sh- Control or LLC-sh-SDCl cells were grown in 10% FBS for 16 hours with SCGB3A2-HT and TMRDirect Halotag ligand. Image was taken under confocal microscope. Bar = 50 pm.

FIGS. llA-llO. SCGB3A2 modeling. (FIGS. 11A-11C) Side-by side comparison of 1UTG (uteroglobin, SCGB1A1) structure (PDBID: 1 UTG) and the model presented herein (SCGB3A2). FIG. 11B shows the helix number and their relative order (2-3- 1-4) similar to what is observed in 1UTG. FIG. 11C shows the relative disposition of the C-terminus, N-terminus, disulfide bridge and the central pocket. The pocket is about half the size of the one observed in 1UTG. Note the very different disposition of the termini and the disulfide bridges in both structures. 1UTG contains two Cys per monomer whereas SCGB3A2 has only one. The disposition of 1UTG disulfide bridges restricts the dimer flexibility controlling the geometry of the pocket mouth. SCGB3A2 shows a much higher flexibility of the corresponding region suggesting a more flexible pocket. (FIG. 11D) 1UTG/SCGB3 A2 sequence alignment obtained from the structural alignment of the two dimers. SCGB3A2 initial model was obtained by aligning the core regions of helices 1 to 4 against the structure of 1UTG. The helix motifs were obtained by analyzing the consensus predictions from several modeling methods. The initial alignment of the predicted helical motifs was performed ignoring the placement of the charged amino acids and cysteines to lower the model bias. After the initial placement was obtained the connecting sections were built as coil sections, and the entire model relaxed using Feedback Restrain Molecular Dynamics (FRMD). The perceived sequence correspondence between 1UTG and SCGB3A2 is presented. Most noticeable albeit not detected in the sequence alignment is the resulting spatial arrangement of SCGB3A2 Cys at the end of the model refinement, placing the cysteines from the monomers in the dimer in a perfect arrangement to satisfy the constraint imposed by a disulfide bridge. The overall sequence functional similarity between 1UTG and SCGB3A2 can be estimated from this alignment at 46% with a similar number of positively charged residues for both structures (11 aa per monomer if His is included) and 11 negatively charged residues per monomer for SCGB3A2 (12 for 1UTG) which may help explain the high propensity of SCGB3A2 to interacting with negatively charged motifs. (FIGS. 11E-11G) General view of the SCGB3A2 dimer rotated 180° in 90° increments to emphasize the placement of the disulfide bridge. The view presented in FIG. 11E is similar to the one previously shown in FIG. 11B. (FIGS. 11H-11J) This view presents the dimer model in a surface decomposition with the charged residues in indicated. The view presented in FIG. 11H is similar to the one previously shown in FIG. 11E and is presented for reference purposes. The model is rotated 180 degrees to expose to faces A and B (FIGS. 1 II and 11J). The anterior (Face A) is dominated by a cluster of positive residues involving Gl (N- termini), R6, K61 and possibly H69 flanking the mouth of the pocket. The posterior orientation (Face B) is dominated by a cluster formed by residues K46, K47, D50, and E51 flanked by D18, D19, K32, and H40. (FIGS. 11K-11M) The relative disposition of the positively charged residues in Face A (FIG. 1 II) corresponds well with a motif capable of a strong interaction with both Heparan and/or LPS showing an ideal separation between the charged motifs of 3.3A (Dl) and 12.5A (D2). (FIG. 11N) A model of a tetramer was explored by rolling a rigid dimer over a second one followed by distance optimization and refinement of the most promising geometries. Putative models for a tetramer were built from the resulting geometries. The most promising arrangements were the result of the mutual avoidance of charges as expected from the highly charged surface of the dimer. The most promising arrangement obtained resulted from the screw rotation of a dimer respect of another with the two interacting by the posterior side of each dimer (Face B, FIG. 11J). This arrangement resulted in the approximate interleaving on charges as indicated by the arrows in bottom right diagram and observed in the 90° rotated view of Face B (bottom center diagram). The bottom left diagram presents a semi-translucent overlapping of the two diagrams to the right (after removal of the annotation) emphasizing the interleaving of the charged residues. The tetramer motif places the two Face A from each dimer far apart minimizing the interaction between the surface positively residues in both dimers. This model suggests the tetrameric form may be capable of simultaneous binding of two negatively charged motifs (i.e. Heparin and LPS). (FIG. 110) SDS-PAGE gel of mSCGB3A2-HT using anti-HaloTag antibody.

FIGS. 12A-12C. Establishment of LLC-sh-TLR4 cells and analysis of SCGB3A2/LPS binding/incorporation. (FIG. 12A) Assessment of shRNA knockdown efficiency in LLC Cells.

LLC cells were transfected with sh-TLR4 plasmid using retrovirus vector and stable cell clones were prepared. Each clone was grown in 2% FBS-RPMI 1640 and total RNA was collected after 48 hours. qPCR analysis of Tlr4 mRNA expression in LLC-sh-Control, and two clones of LLC-sh- TLR4. sh-TLR4(A) clone was used in further assays. (FIG. 12B) Immunofluorescent staining for LPS_A594 binding to LLC cells. LLC-sh-Control, LLC-sh-SDCl, or LLC-sh-TLR4 cells were incubated with LPS_A594 (2 pg/ml) with or without SCGB3A2 (4 pg/ml) in 0% FBS-RPMI 1640 for 3 hours. Nuclei were stained with DAPI. Images are the representatives of three independent experiments. Bar = 10 pm. (FIG. 12C) Live cell imaging using SCGB3A2-HT supernatant, Halotag ligand, and LPS_A488 of LLC-sh-TLR4 cells. Cells were cultured in 1% FBS-RPMI 1640 for 16 hours. Halotag signals conjugated with SCGB3A2 were visualized with TMRDirect HaloTag ligand. Images are the representatives of three independent experiments. S2: SCGB3A2. Bar = 10 pm.

FIGS. 13A-13D. Effect of SCGB3A2-LPS on RAW264.7 cells. (FIG. 13A) Flow cytometric analysis for SDC1 expression on RAW264.7 cells. PE-conjugated mouse SDC1 ectodomain specific antibody was used for detection of cell surface SDC1 expressions on

RAW264.7 cells. (FIG. 13B) PMb secretion into culture medium determined by ELISA. RAW 264.7 cells treated with human SCGB3A2 (1 pg/ml), LPS (Ol ll:B4) (1 pg/ml), and/or heparin (1 pg/ml) were cultured in OPTI-MEM for 16 hours and supernatant was harvested and analyzed.

Data are the representative of three independent experiments. ND: not detectable. (FIG. 13C) LDH (lactate dehydrogenase) release from unprimed RAW264.7 cells with or without LPS (1 pg/ml) and/or SCGB3A2 (1 pg/ml) for 16 hours in OPTI-MEM. Data are the representative of three independent experiments, each carried out in triplicate. S2: SCGB3A2, L+S: LPS+SCGB3A2. **: p<0.0l by one-way ANOVA. (FIG. 13D) Immunofluorescence analysis of RAW264.7 cells using LPSA5₉₄, anti-caspase-l l, or anti-NLRP3 antibodies. Cells were treated with or without LPSA5₉₄ (1 pg/ml) and/or human SCGB3A2 (1 pg/ml) in 1% FBS-RPMI 1640 for 16 hours. Arrowheads indicate caspase-l l foci. Bar=l0pm.

FIGS. 14A-14T. Immunofluorescence analysis of human cell lines for SDC1 and HS. (FIGS. 14A-14T) Representative images of immunofluorescence analysis for human SDC1 and HS. Nucleus was counterstained with DAPI. All cells were incubated in 10% FBS-RPMI for 24 hours. Floating cells (FIGS. 14G, 14J, 14M, 14P, 14S, and 14T) were cytospin preparations. All other cells were cultured in chamber slide. Bar = 10 pm. (Right column) Relative CASP4 mRNA level of each cell line incubated in 10% FBS-RPMI for 24 hours. A549 mRNA level was set as 1.00.

FIGS. 15A-15T. Flow cytometry analysis of human cell lines for SDC1 and HS expression. Flow cytometry analysis of SDC1 and HS expressions on cell surfaces of human cancer cell lines. Gray histograms indicate the unstained control samples. Calculated mean and median values in each sample are indicated in Table 3. Graphs are representative of three independent experiments.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on January 10, 2019, 12.3 KB, which is incorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is an amino acid sequence of mature human SCGB3A2 protein.

FLINKVPLPVDKLAPLPLDNI LPFMDPLKLLLKTLGI SVEHLVEGLRKCVNELGPEASEAVKKLLE

ALSHLV

SEQ ID NO: 2 is an amino acid sequence of human SCGB3 A2 precursor protein (NCBI Reference Sequence: NP_473364.l, incorporated by reference herein).

MKLVTIFLLVTISLCSYSATAFLINKVPLPVDKLAPLPLDNILPFMDPLKLLLKTLGISVEHLVEG LRKCVNELGPEASEAVKKLLEALSHLV

SEQ ID NO: 3 is an exemplary nucleotide sequence encoding human SCGB3A2 precursor protein (NCBI Reference Sequence: NM_054023.4, incorporated by reference herein).

atgaagctggtaactatcttcctgctggtgaccatcagcctttgtagttactctgctactgccttc ctcatcaacaaagtgccccttcctgttgacaagttggcacctttacctctggacaacattcttccc tttatggatccattaaagcttcttctgaaaactctgggcatttctgttgagcaccttgtggagggg ctaaggaagtgtgtaaatgagctgggaccagaggcttctgaagctgtgaagaaactgctggaggcg ctatcacacttggtgtga

SEQ ID NO: 4 is an amino acid sequence of mature mouse SCGB3A2 protein.

LLINRLPWDKLPVPLDDI IPSFDPLKMLLKTLGI SVEHLVTGLKKCVDELGPEASEAVKKLLEAL

SHLV

SEQ ID NO: 5 is an amino acid sequence of mouse SCGB3A2 precursor protein (NCBI Reference Sequence: NP_00l276573.l, incorporated by reference herein).

MKLVSIFLLVTIGICGYSATALLINRLPWDKLPVPLDDIIPSFDPLKMLLKTLGISVEHLVTGLK

KCVDELGPEASEAVKKLLEALSHLV

SEQ ID NO: 6 is an exemplary nucleotide sequence encoding mouse SCGB3A2 precursor protein (NCBI Reference Sequence: NM_001289644.1, incorporated by reference herein).

atgaagctggtatctatctttctgctggtgaccattggtatttgtggttattctgccactgccctt ctcatcaaccgtctccctgttgttgacaaattacctgtacctttggacgacattattccctcattt gatcccttgaagatgcttctgaaaaccctgggcatttctgtagaacatctggtgacaggactgaag aagtgtgtggacgagctgggaccagaggcttccgaggccgtgaagaagcttctggaggctctttca cacctggtataa

SEQ ID NO: 7-15 are shRNA sequences used for mouse Sdcl knock down.

SEQ ID NO: 16-17 are shRNA sequences used for mouse TLR4 knock down.

SEQ ID NO: 18-41 are primer sequences used for real-time PCR. SEQ ID NO: 42 is an SCBG3A2 amino acid sequence (see FIG. 11D).

SEQ ID NO: 43 is a 1UTG amino acid sequence (see FIG. 11D).

DETAILED DESCRIPTION

Lipopoly saccharide (LPS) is a component of the outer membrane of gram negative bacteria and can cause inflammation in the lung. It was previously thought that toll-like receptor 4 (TLR4) was the sole LPS-specific pattern recognition receptor (PRR) at the cell membrane (Poltorak et al, Science, 282, 2085-2088, 1998). Further studies demonstrated the presence of a TLR4-independent PRR mechanism to sense LPS in the cytosol via an inflammatory caspase, caspase- 11 (in mice) or caspase 4/5 (in humans), via a non-canonical inflammasome pathway. Activation of the non- canonical inflammasome leads to caspase- 1 activation, the production of pro-inflammatory cytokines such as IL-l and IL-18, and inflammatory cell death by“pyroptosis”. Pyroptosis is characterized by the appearance of membrane pores, cell swelling followed by membrane rupture, and release of the intracellular contents including the aforementioned IL-l and IL-18, and lactate dehydrogenase (LDH). However, the mechanism for activation of the non-canonical

inflammasome pathway, in which caspase- 11 participates, was not completely understood, especially in cell types other than immune cells.

Secretoglobin 3A2 (SCGB3A2) is a small secretory protein expressed in lung airway epithelial cells that was previously shown to suppress pulmonary inflammation and fibrosis.

This disclosure shows that SCGB3A2 is an LPS binding protein and that the SCGB3A2- LPS complex binds to a receptor, SDC1, on the cell surface of cancer cells, after which it is internalized and induces pyroptotic cell death via the non-canonical inflammasome. LPS and SCGB3A2 co-localize into uropod-like structures, which overlap with the polarized expression of SDC1 and clathrin, the protein critical for intracellular trafficking. These results indicate that the SCGB3A2-LPS complex, via binding to SDC1, is incorporated in cancer cells through clathrin- mediated endocytosis, resulting in upregulation of a non-canonical inflammasome pathway and pyroptotic death.

Further, data is provided herein showing that, in a lung carcinoma cell murine tumor model, SCGB3A2 and LPS possessed potent anti-cancer activity by stimulating the non-canonical inflammasome pathway driven by caspase- 11/NLRP3 activation and pyroptotic cell death of the lung carcinoma cells. Several human lung and colorectal carcinoma cells also showed strong susceptibility to SCGB3A2-induced growth inhibition and expressed abundant SDC1 on the cell surface and CASP4 mRNA expression. Thus, based on prior results, SCGB3A2 was believed to be an anti-inflammatory molecule. Surprisingly, based on the results disclosed herein, SCGBA2 also facilitates“inflammation-induced death” of cells, such as of cancer cells, when in the presence of LPS and when the cells have cell- surface expression of SDC1 and express non-canonical inflammasome components, such as caspase 4 and caspase 5.

I. Terms

Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017.

The singular terms“a”,“an”, and“the” include plural referents unless context clearly indicates otherwise. The term“comprises” means“includes.” Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. In case of conflict, the present specification, including terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. In order to facilitate review of the various embodiments of the disclosure, the following explanation of terms is provided:

About: With reference to a numerical parameter, the term“about” refers to a plus or minus 5% range around the numerical parameter. For example,“about 5%” refers to“4.75% to 5.25%.”

Administration: To provide or give to a subject an agent, for example, a composition comprising SCGB3A2 protein and LPS, by any effective route. Administration can be local or systemic. Exemplary routes of administration include, but are not limited to, oral (for example, oral administration of a composition comprising SCGB3A2 and LPS that delays release of the SCGB3A2 and LPS until the composition is in the intestine, such as the colon), injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, direct injection into intestine (for example, injection into the colon)), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation (particularly in the case of a treatment for lung cancer) routes.

Cancer: A malignant neoplasm that has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and is capable of metastasis. Non-limiting examples of cancers include sarcomas (connective tissue cancer) and carcinomas (epithelial cell cancer), include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

Residual cancer is cancer that remains in a subject after any form of treatment given to the subject to reduce or eradicate the cancer. Metastatic cancer is a tumor at one or more sites in the body other than the site of origin of the original (primary) cancer from which the metastatic cancer is derived. Cancer includes, but is not limited to, solid tumors.

Carcinoma: A malignant tumor including transformed epithelial cells. Non-limiting examples of carcinomas include adenocarcinoma, squamous cell carcinoma, anaplastic carcinoma and large and small cell carcinoma. In some examples, a carcinoma is a colorectal carcinoma or lung carcinoma.

Caspase 4: A cytosolic cysteine protease that cleaves proteins at an aspartic acid residue. LPS binding to caspase 4 can activate the non-canonical inflammasome to induce pyroptosis. Caspase 4 protein is encoded by the CASP4 gene (NCBI Gene ID No. 837). An exemplary protein sequence for human caspase 4 is set forth as NCBI reference sequence NR_001216.1 (accessed January 3, 2018, incorporated by reference herein). An exemplary encoding sequence for human caspase 4 is set forth as NCBI reference sequence NM_00l225.3 (accessed January 3, 2018, incorporated by reference herein).

Caspase 5: A cytosolic cysteine protease that cleaves proteins at an aspartic acid residue. LPS binding to caspase 5 can activate the non-canonical inflammasome to induce pyroptosis. Caspase 5 protein is encoded by the CASP5 gene (NCBI Gene ID No. 838). An exemplary protein sequence for human caspase 5 is set forth as NCBI reference sequence NP_00l 129581.1 (accessed January 3, 2018, incorporated by reference herein). An exemplary encoding sequence for human caspase 5 is set forth as NCBI reference sequence NM_00l 136109.2 (accessed January 3, 2018, incorporated by reference herein). Caspase 11: A cytosolic cysteine protease in mice that cleaves proteins at an aspartic acid residue. LPS binding to caspase 11 can activate the non-canonical inflammasome to induce pyroptosis. Caspase 11 protein is encoded by the Caspll (also known as Scafll ) gene (NCBI Gene ID No. 72193). An exemplary protein sequence for mouse caspase 11 is set forth as NCBI reference sequence NP_082424.2 (accessed January 3, 2018, incorporated by reference herein). An exemplary encoding sequence for mouse caspase 11 is set forth as NCBI reference sequence NM_028l48.2 (accessed January 3, 2018, incorporated by reference herein).

Chemotherapeutic agents: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth such as psoriasis.

In one embodiment, a chemotherapeutic agent is an agent of use in treating a SDC1 positive cancer, such as SDC1 positive lung or colorectal cancer. Non- limiting examples of chemotherapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors.

Colorectal cancer: A neoplastic tumor of colon, rectum or anus tissue that is or has the potential to be malignant. The main types of colorectal cancer include colorectal carcinomas such as adenocarcinoma and squamous cell carcinoma. Infiltrating (malignant) carcinoma of the colon can be divided into stages (I, II, III and IV). See, for example, Blake et al. (eds.), Gastrointestinal Oncology: A practical Guide, Berlin: Springer- Verlag, 2011.

Conservative amino acid substitution: An amino acid substitution that does not substantially affect the function of a protein.

For example, a conservative amino acid substitution in SCGB3A2 is one that does not reduce the LPS binding and syndecan-l binding of SCGB3A2 by more than 10% (such as by more than 5%) compared to the LPS binding and syndecan-l binding of a parent SCGB3A2 protein (such as SCGB3A2 protein set forth as SEQ ID NO: 1). SCGB3A2 binding to LPS and syndecan-l can be measured using methods described herein, as well as those available in the art. Another approach for determining if an amino acid substitution in SCGB3A2 is a conservative amino acid substitution is to assess the anticancer activity of the SCGB3A2 including the amino acid substitution. For example, a conservative amino acid substitution is one that does not reduce the anti-cancer activity of SCGB3A2 and LPS by more than 10% (such as by more than 5%) compared to the anti-cancer activity of a parent SCGB3A2 protein (such as SCGB3A2 protein set forth as SEQ ID NO: 1). The anti-cancer activity of SCGB3A2 and LPS can be measured using methods described herein, as well as those available in the art. In some embodiments, the anti-cancer activity is measured using the LLC metastasis model as described in the Examples.

The following six groups are examples of amino acids that are considered to be

conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Contacting: Placement in direct physical association, for example solid, liquid or gaseous forms. Contacting includes, for example, direct physical association of fully- and partially-solvated molecules.

Decrease or Reduce: To reduce the quality, amount, or strength of something; for example a reduction in tumor burden. In one example, a treatment reduces a tumor (such as the size of a tumor, the number of tumors, the metastasis of a tumor, or combinations thereof), or one or more symptoms associated with a tumor (such as pathological angiogenesis of the tumor or tumors), for example as compared to the response in the absence of the therapy. In a particular example, a treatment decreases the size of a tumor, the number of tumors, the metastasis of a tumor, or combinations thereof, subsequent to the therapy, such as a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

Such decreases can be measured, for example, using conventional methods, as well as the methods disclosed herein.

Detecting expression of a gene product: Determining the presence of and/or the level of expression of a nucleic acid molecule (such as an mRNA molecule) or a protein encoded by a gene in either a qualitative or quantitative manner. Exemplary methods include microarray analysis, RT- PCR, Northern blot, Western blot, and mass spectrometry of specimens from a subject, for example measuring levels of a gene product present in blood, serum, or another biological sample as a measure of expression.

Endotoxin Unit (EU): Endotoxin levels are often measured in EU/mL as determined by limulus amebocyte lysate (LAL) assay, for example, as described in the United States

Pharmacopeia and National Formulary (USP 35-NF 30) chapter <85> (Rockville, MD: United States Pharmacopeial Convention; 2012). One EU is equivalent to approximately 100 pg of LPS. Expression: The process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of a protein. Gene expression can be influenced by external signals. Different types of cells can respond differently to an identical signal. Expression of a gene also can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

Controls or standards for comparison to a sample, for the determination of differential expression, include (but are not limited to) samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from cells or tissue that does not express the non-canonical inflammasome) or a sample of cells or tissue known to have or to not have cell- surface expression of SDC1, as well as laboratory values (e.g., range of values), even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory. Laboratory standards and values can be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured,

experimentally determined values.

Lipopolysaccharide (LPS): Also known as lipoglycan and endotoxin, LPS is a major constituent of the outer membrane of the wall of Gram-negative bacteria. LPS is toxic at high doses to mammals. It is responsible for septic shock, a fatal pathology which develops following acute infection with a Gram-negative bacterium.

LPS structure includes a lipid portion, called lipid A, covalently bonded to a polysaccharide portion. Lipid A is responsible for the toxicity of LPS. It is highly hydrophobic and enables the LPS to be anchored in the outer membrane of the bacterial cell wall. Lipid A is composed of a disaccharide structure substituted with fatty acid chains. The number and the composition of the fatty acid chains varies between bacterial species. At least 3 major regions can be distinguished in the polysaccharide portion: (i) an inner core composed of monosaccharides [one or more KDO (2- keto-3-deoxyoctulosonic acid) and one or more heptose (Hep) moieties] which do not change within the same bacterial species; (ii) an outer core bonded to the heptose and composed of various monosaccharides; and (iii) an O-specific outer chain composed of a series of repeating units of one or more different monosaccharides. The composition of the polysaccharide portion varies from one species to another, from one serotype (immunotype in meningococcus) to another within the same species. As used herein, the term“LPS” is intended generally. The LPS used in the disclosed methods and compositions can be from any suitable Gram-negative bacteria. In some

embodiments, the LPS is from one of Escherichia coli 011 LB4, E. coli K-235, Salmonella typhimurium, or a Ra mutant LPS from E. coli EH-100. Additional sources of LPS include, but are not limited to, E. coli 0111.B8, E. coli 0127:B8, E. coli 0128:B12, E. coli 026.B6, E. coli 055.B5, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica, Salmonella typhosa, Serratia marcesens, E. coli 0157:H7, Enterrobacter cloacae, Heliobacter pyroli, Klebsiella pneumoniae, Haemophilus influenza.

Lung cancer: A neoplastic tumor of lung tissue that is or has the potential to be malignant. The main types of tumors of the lung are lung carcinomas: adenocarcinoma, small cell carcinoma, squamous cell carcinoma, large cell carcinoma, or non-small cell carcinoma. Standardized lung cancer classifications by the World Health Organization have traditionally been based on the histological characteristics of resected tumors with little guidance about diagnosis based on small biopsies and cytology. The focus has mainly been on the separation of small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), which includes adenocarcinoma (AD), squamous cell carcinoma (SCC), large cell carcinoma (LC) and bronchioalveolar carcinoma (BAC). Both AD and LC are classified as non-squamous cell type carcinoma. Lung cancer is typically staged from I to IV; other classifications are also used, for example small-cell lung carcinoma can be classified as limited stage if it is confined to one half of the chest and within the scope of a single radiotherapy field; otherwise, it is extensive stage. See, for example, Hansen (ed.), Textbook of Lung Cancer, 2^nd, London: Informa Healthcare, 2008.

Normal cells or tissue: Non-tumor, non-malignant cells and tissue, such as lung tissue.

Nanoparticles: Solid colloidal particles that range in size from about 10-1000 nm. They can be made from biodegradable and biocompatible biomaterials. Active components, such as drugs, can be adsorbed, encapsulated, or covalently attached to their surface or into their matrix.

Non-canonical inflammasome: A cytosolic multi-protein complex that regulates the secretion of proinflammatory cytokines to induce pyroptosis. Activation of the non-canonical inflammasome is triggered by cytosolic sensing of LPS. This pathway is independent of Toll-like receptor 4 (TLR4), the well-known extracellular receptor for LPS, but instead depends on LPS- binding by inflammatory proteases caspase-4 and/or caspase-5 in humans, or caspase 11 in mice. The non-canonical inflammasome is described, for example, in Man and Kanneganti,“Converging roles of caspases in inflammasome activation, cell death and innate immunity,” Nat Rev

Immunol. , 16(1):7 -21, 2016; Zhao et a ,“NLRP3 inflammasome activation plays a carcinogenic role through effector cytokine IL-18 in lymphoma,” Oncotarget., 8(65), 2017; Ablasser and Dorhoi,“Tnflammasome Activation and Function During Infection with Mycobacterium

Tuberculosis,” Curr Top Microbiol Immunol., 397: 183-97, 2016; Crowley et al.,“Noncanonical inflammasomes: Antimicrobial defense that does not play by the mles,” Cell Microbiol., 19(4) 2017; Place and Kanneganti,“Recent advances in inflammasome biology,” Curr Opin Immunol., 50:32-38, 2017; and Liu and Lieberman,“A Mechanistic Understanding of Pyroptosis: The Fiery Death Triggered by Invasive Infection,” Adv Immunol., 135:81-117, 2017

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22^nd ed. , London, UK:

Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g. , powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non- natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some embodiments, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill or in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed). Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms

“polypeptide” or“protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. A polypeptide includes both naturally occurring proteins, as well as those that are recombinantly or synthetically produced. A polypeptide has an amino terminal (N-terminal) end and a carboxy-terminal (C-terminal) end.

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein (such as an SCGB3A2 protein) is more enriched than the protein is in its natural environment within a cell. In one embodiment, a preparation is purified such that the protein represents at least 50% of the total protein content of the preparation.

Promoter: An array of nucleic acid control sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Promoters may be constitutive or inducible.

SCGB3A2: Also called uteroglobin-related protein 1 (UGRP1), LuLeul, lul03, Pnspl, and Hin-2, SCGB3A2 is a member of the uteroglobin/Club cell secretory protein (UG/CCSP) gene superfamily of secretory proteins, which share a common four helical bundle subunit structure, exist as dimers, tetramers, and other oligomers. SCGB3A2 is predominantly expressed in the epithelial cells of trachea, bronchus, and bronchioles. SCGB3A2 has been shown to suppress lung inflammation using a mouse model for allergic airway inflammation (Yonede et al, Int. Arch. Allergy Immumol., 171(1): 36-44, 2016; Chiba el al, (2006) Am. J. Respir. Crit. Care Med May l:l73(9):958-64.). MARCO, a macrophage scavenger receptor that is expressed in lung alveolar macrophages and is involved in pulmonary inflammation, has been identified as the receptor for SCGB3A2 (Bin et al , (2003) J. Immunol. 171, 924-30).

As used herein, the term“SCGB3A2” is intended generally. Thus, in one embodiment, SCGB3A2 is a human protein. In another embodiment, SCGB3A2 is a non-human animal homolog/ortholog of the human molecule, such as a sheep, chimpanzee, goat, pig, mouse, rat, or hamster SCGB3A2-equivalent protein. The term SCGB3A2 includes variant SCGB3A2 proteins having at least 90% sequence identity to a native SCGB3A2. As used herein, the term“SCGB3A2 without LPS” refers to a composition of SCGB3A2 that contains less than 0.01 EU/pg LPS.

SDC1: A transmembrane heparan sulfate proteoglycan and member of the syndecan proteoglycan family. Syndecans mediate cell binding, cell signaling and cyoskelatal organization. The human SDC1 protein has UniProt ID P18827. The GenBank Accession number for human syndecan-l protein is NP_001006947.1 (accessed January 3, 2018). The GenBank Accession number for human syndecan-l coding sequence is NM_001006946.1 (accessed January 3, 2018).

SDC1 positive cancer: A cancer with cells having surface expression of SDC1. Any appropriate technique can be used to determine if the cells of cancer have surface expression of SDC1, such as immunohistochemistry assays, immunofluorescence analysis, flow cytometric analysis, ELISA, dot blotting. In some examples, an SDC1 positive cancer is one with detectable levels of cell surface SDC1 relative to a control as determined using an

immunohistochemistry/immunofluorescence/flow cytometric assays. In another embodiment, an SDC1 positive cancer is one where at least 5% (such as at least 10%, at least 20 %, at least 30%, at least 40%, at least 50%, at least 75%, or at least 80%) of the cells in a sample from the cancer have cell-surface expression of SDC1 as detected by immunohistochemistry, immunofluorescence, or flow cytometry. Non-limiting examples of cancers that may be SDC1 positive include lung and colorectal cancers.

Sequence identity: The similarity between amino acid and nucleotide sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2(4):482-489, 1981; Needleman and Wunsch, J. Mol. Biol. 48(3):443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85(8):2444-2448, 1988; Higgins and Sharp, Gene,

73(l):237-244, 1988; Higgins and Sharp, Bioinformatics, 5(2): 151-3, 1989; Corpet, Nucleic Acids Res. 16(22):10881-10890, 1988; Huang et al. Bioinformatics, 8(2): 155-165, 1992; and Pearson, Methods Mol. Biol. 24:307-331, 1994. Altschul et al, J. Mol. Biol. 215(3):403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215(3):403-410, 1990) is available from several sources, including the National Center for Biological Information and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100.

Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods. Homologs and variants of a polypeptide are typically characterized by possession of at least about 80%, for example at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity counted over the full length alignment with the amino acid sequence of interest.

Subject: Any mammal, such as humans, non-human primates, pigs, sheep, cows, rodents, and the like. In two non-limiting examples, a subject is a human subject or a murine subject. Thus, the term“subject” includes both human and veterinary subjects.

Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or suppress growth of a tumor. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor. For example, the agent or agents can decrease the size, volume, or number of tumors by a desired amount, for example by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 90%, or at least 95% as compared to a response in the absence of the agent. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.

Treating or preventing a disease: “Preventing” a disease refers to inhibiting the full development of a disease, for example in a person who is known to have a predisposition to a disease such as a cancer.“Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.“Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer. In several embodiments, treatment refers to a reduction in size of a tumor, a decrease in the number and/or size of metastases, or a decrease in a symptom of the tumor. Tumor: An abnormal mass of tissue resulting from excessive cell division that is uncontrolled and progressive, also called a neoplasm. Tumors of the same tissue type are primary tumors originating in a particular organ (such as breast, prostate, bladder or lung). Tumors of the same tissue type may be divided into tumor of different sub-types (a classic example being bronchogenic carcinomas (lung tumors) which can be an adenocarcinoma, small cell, squamous cell, or large cell tumor). A tumor that does not metastasize is referred to as“benign.” A tumor that invades the surrounding tissue or can metastasize (or both) is referred to as“malignant.” “Metastatic disease” refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system. A“tumor cell” is a neoplastic cell derived from a tumor. A tumor originating from a particular tissue can be referred to by that tissue, e.g., a tumor originating from lung tissue can be referred to as a“lung tumor.” Several embodiments include treatment of a lung tumor. Tumor proliferation is abnormal growth due to cell division.

Tumor burden: The total volume, number, metastasis, or combinations thereof of tumor or tumors in a subject, or in an organ of a subject.

Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity. In one example the desired activity is treatment of a tumor.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transfected host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.

II. SCGB3A2 and LPS

The SCGB3A2 protein for use in the disclosed methods and compositions can have a sequence from any appropriate source, such as human SCGB3A2, or a non-human animal homolog/ortholog, such as a sheep, chimpanzee, goat, pig, mouse, rat, or hamster SCGB3A2- equivalent protein.

In some embodiments, the SCGB3A2 protein for use in the disclosed methods and compositions comprises or consists of an amino acid sequence set forth as SEQ ID NO: 1, or a sequence at least 90% (such as at least 95%, or at least 98%) identical to SEQ ID NO: 1 that binds to SDC1 and LPS. In some embodiments, the SCGB3A2 protein for use in the disclosed methods and compositions has no more than 10 (such as no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions (such as conservative amino acid substitutions) compared to SEQ ID NO: 1.

Manipulation of the nucleotide sequence of SCGB3A2 using standard procedures, including in one specific, non-limiting, embodiment, site-directed mutagenesis or in another specific, non-limiting, embodiment, PCR, can be used to produce such variants. The simplest modifications involve the substitution of one or more amino acids for amino acids having similar biochemical properties. These so-called conservative substitutions are likely to have minimal impact on the activity of the resultant protein.

Methods of producing and purifying SCGB3A2 protein and nucleic acid molecules encoding SCGB3A2 protein are described herein and known in the art (see, e.g., Kurotani et al., J. Biol. Chem., 286:19682-19692, 2011; Kurotani et al, Am. J. Respir. Crit. Care. Med., 178:389- 398, 2008; J. Biol. Chem., 286:19682-19692, 2011; Niimi et al, Mol. Endocrinol., 15:2021-36, 2001; Int. Pub. U.S. App. W02008/039941; and U.S. Pub. No. US20140274915, each of which is incorporated by reference herein in its entirety).

The human SCGB3A2 gene is about 2,900 base pairs in length and consists of three exons. The first intron of SCGB3A2 is about five to six- fold longer than the second intron, which resembles the structure of orthologous mouse Scgb3a2 gene. Non-limiting examples of SCGB3A2 nucleotide sequences include the following GENBANK™ accession numbers for murine

SCGB3A2/UGRP1 (Nos. AF274959.1, AF274960.1, AF274961.1) and human SCGB3A2/UGRP1 (No. AF313455.1). The above accession numbers are incorporated by reference herein as present in the GENBANK™ database on January 3, 2018. These sequences are provided merely as examples; other proteins/nucleic acids that fall into the described class will be recognized. These exemplary SCGB3A2 nucleic acid sequences can be used to produce SCGB3A2 protein, for example by introducing a SCGB3A2 encoding sequence into an appropriate vector, transducing cells with the vector for protein expression, and purifying the expressed SCGB3A2.

The LPS used in the disclosed methods and compositions can be LPS from any suitable Gram-negative bacteria. In some embodiments, the LPS is from one of Escherichia coli Oll l:B4 (E4391), E. coli K-235 (L2018), Salmonella typhimurium (L2262), or a Ra mutant LPS from E. coli EH-100 (L9641). Additional sources of LPS include, but are not limited to, E. coli 0111.B8, E. coli 0127 :B8, E. coli 0128:B12, E. coli 026.B6, E. coli 055.B5, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica, Salmonella typhosa, Serratia marcesens, E. coli 0157:H7, Enterrobacter cloacae, Heliobacter pyroli, Klebsiella pneumoniae, Haemophilus influenza. Suitable forms of LPS can be purchased commercially (e.g., from MilliporeSigma), or produced using standard techniques. Non- limiting examples of LPS preparation methods include trichloroacetic acid (TCA) extraction, phenol extraction, and phenolchloroform-petroleum ether extraction. TCA- and phenol- based extranction methods are commonly used to purify LPS. The TCA extracted

lipopolysaccharides are structurally similar to the phenol extracted ones. Their electrophoretic pattern and endotoxicity are similar. The main differences are in the amounts of nucleic acid and protein contaminations. The TCA extract contains approximately 2% RNA and approximately 10% denatured proteins. The phenol extract contains up to 60% RNA and less than 1% protein. Further purification by gel filtration chromatography removes much of protein present in the phenol- extracted LPS, but leaves a product that still contains 10-20% nucleic acids. Further purification using ion exchange chromatography, yields an LPS product which contains <1% protein and <1% RNA.

III. Treatment and Prevention of Cancer

It is shown herein that administration of SCGB3A2 and LPS inhibits the growth and metastasis of tumors in vivo. This observation supports the use of SCGB3A2 in combination with LPS as a therapeutic for the treatment of cancer.

Accordingly, methods are disclosed herein for treating cancer (such as lung or colorectal cancer) in a subject by administrating a therapeutically effective amount of SCGB3A2 and LPS to the subject.

In additional embodiments, methods are provided for treating cancer (such as colorectal cancer) in a subject by administrating a therapeutically effective amount of SCGB3A2 without LPS to the subject, wherein the cancer is not lung cancer. In such embodiments, the SCGB3A2 is administered to a target location in the subject (such as local administration to a tumor) that already has a sufficient concentration of LPS present to facilitate SCGB3A2 cellular entry into the cells of the tumor.

In some embodiments, the methods include treating an existing cancer (such as lung or colorectal cancer) in a subject. In additional embodiments, methods are disclosed herein are used for preventing metastasis of a cancer in a subject. The cancer can be benign or malignant.

Subjects that can benefit from the disclosed methods include humans and veterinary subjects. A suitable administration format may be determined by a medical practitioner for each subject individually. Various pharmaceutically acceptable carriers and their formulation are known. The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. Subjects can be screened prior to initiating the disclosed therapies, for example to determine whether the subject has cancer, such as an epithelial cell cancer (/._<?., carcinoma), and/or a lung or colorectal cancer. In some embodiments, the epithelial cell cancer is a cancer of epithelial tissue that is naturally exposed to exogenous microorganisms, such as cancers originated from

nasopharynx, larynx, cervix, lung, or colon epithelial tissue. The presence of the cancer in the subject indicates that the cancer can be treated using the methods provided herein. The presence of a cancer in a subject can be determined by methods known in the art, and typically include cytological and morphological evaluation. The cancer can be one with an established tumor. The cells of the cancer that are screened can be in vivo or ex vivo, including cells obtained from a biopsy. In some embodiments, a subject can be selected for treatment that has, is suspected of having, or is at risk of developing, cancer, such as lung or colorectal cancer.

In some embodiments, the cancer is a SDC1 positive cancer. Any suitable technique can be used to determine if the cells of a cancer in a subject are SDC1 positive. In a non-limiting example, the SDC1 positive cancer is identified by conducting an

immunohistochemistry/immunofluorescence/flow cytometric assay on a patient biopsy. Non limiting examples of cancers that may be SDC1 positive include lung and colorectal cancers.

In some embodiments, the cancer is positive for expression of genes of the non-canonical inflammasome. For example, the cancer comprises cells expressing CASP4 and/or CASP5. Any suitable technique can be used to determine if the cells of a cancer in a subject are positive for expression of genes of the non-canonical inflammasome, such as CASP4 and/or CASP5. In a non limiting example, the expression of genes of the non-canonical inflammasome, such as CASP4 and/or CASP5, is identified by conducting an RT-PCR assay for mRNA levels of genes of the non- canonical inflammasome, such as CASP4 and/or CASP5, on a patient biopsy.

In a preferred embodiment, the cancer is an SDC1 positive cancer, and is also positive for expression of genes of the non-canonical inflammasome, such as CASP4 and/or CASP5

The cancer treated by the methods disclosed herein can be any cancer of interest, including, but not limited to, lung or colorectal cancer. Non-limiting examples of cancers that can be treated using the disclosed methods include skin cancers, breast cancers, brain cancers, cervical carcinomas, testicular carcinomas, head and neck cancers, gastrointestinal tract cancers, genitourinary system cancers, gynecological system cancers, endocrine system cancers, a sarcoma of the soft tissue and bone, a mesothelioma, a melanoma, a neoplasm of the central nervous system, and a leukemia. In some embodiments, the cancer is a head and neck cancer, such as cancers of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands and paragangliomas. In other embodiments, the cancer is a lung cancer, such as a non-small cell lung cancer (NSCLC) or a small cell lung cancer. In some examples, the NSCLC is adenocarcinoma, squamous cell carcinoma, large cell carcinoma or bronchioalveolar carcinoma.

In further embodiments, the cancer can be a cancer of the gastrointestinal tract, such as cancer of the esophagus, stomach, pancreas, liver, biliary tree, small intestine, colon, rectum and anal region. In yet other embodiments, the cancer can be a cancer of the genitourinary system, such as cancer of the kidney, urethra, bladder, prostate, urethra, penis and testis. In some embodiments, the cancer is a gynecologic cancer, such as cancer of the cervix, vagina, vulva, uterine body, gestational trophoblastic diseases, ovarian, fallopian tube, peritoneal, or breast. In other embodiments, the cancer is an endocrine system cancer, such as a thyroid cancer, parathyroid cancer, adrenal cortex cancer, pancreatic endocrine cancer, carcinoid cancer and carcinoid syndrome. The cancer can be a sarcoma of the soft tissue and bone, a mesothelioma, a cancer of the skin, a melanoma, comprising cutaneous melanomas and intraocular melanomas, a neoplasm of the central nervous system, a cancer of the childhood, comprising retinoblastoma, Wilm's tumor, neurofibromatoses, neuroblastoma, Ewing's sarcoma family of cancers, rhabdomyosarcoma. The cancer can be a lymphoma, comprising non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, primary central nervous system lymphoma, and Hodgkin's disease. The cancer can be a leukemia, such as acute leukemia, chronic myelogenous leukemia and lymphocytic leukemia. The cancer can be plasma cell neoplasms, a cancer of unknown primary site, a peritoneal carcinomastosis, a Kaposi's sarcoma, AIDS-associated lymphomas, AIDS-associated primary central nervous system lymphoma, AIDS-associated Hodgkin's disease and AIDS-associated anogenital cancers, a metastatic cancer to the liver, metastatic cancer to the bone, malignant pleural and pericardial effusions and malignant ascites. In specific non-liming examples, the cancer is melanoma or colon cancer, or a cancer that has metastasized to the lung (such as a colon cancer or a breast cancer that has metastasized to the lung).

In one embodiment, the disclosed methods are used to treat lung cancer in a subject.

Treatment of the lung cancer can reduce a symptom of a lung cancer in the subject. Symptoms include respiratory symptoms, such as coughing, coughing up blood, wheezing and/or shortness of breath, systemic symptoms such as weight loss, fever, or fatigue, or symptoms due to local compression, such as chest pain, bone pain, or difficulty swallowing. In some examples, the lung cancer is SCLC or NSCLC. The non-small cell lung cancer can be squamous cell lung carcinoma, adenocarcinoma (ADC), and large cell lung carcinoma. Generally, the methods include selecting a subject having a lung cancer, such as a NSCLC or a SCLC, and administering to the subject a therapeutically effective amount of a SCGB3A2 and LPS. Treatment of the lung cancer, such as NSCLC, is generally initiated after the diagnosis of the lung cancer. A subject with any stage of lung cancer can be treated using the method disclosed herein. The presence of lung cancer, such as NCSLC, can be determined by methods known in the art, such as a CT scan, a PET scan, endoscopic ultrasound and/or endobronchial ultrasound. Pulmonary function tests can also be used. The lung cancer can also be diagnosed by obtaining one or more biopsies and evaluating the cells in the biopsy.

In one embodiment, the disclosed methods are used to treat a colorectal cancer in a subject. Treatment of the colorectal cancer can reduce a symptom of a colorectal cancer in the subject. Typical symptoms include abdominal pain and change in bowel habits, as well as systemic symptoms such as weight loss, fever, and fatigue. In some examples, the colorectal cancer is an adenocarcinoma, a carcinoid tumor, or a gastrointestinal stromal tumor. Generally, the methods include selecting a subject having a colorectal cancer, such as an adenocarcinoma, and

administering to the subject a therapeutically effective amount of SCGB3A2 without LPS, or SCGB3A2 and LPS. Treatment of the colorectal cancer, such as an adenocarcinoma, is generally initiated after the diagnosis of the colorectal cancer. A subject with any stage of colorectal cancer can be treated using the method disclosed herein. The presence of colorectal cancer, such as an adenocarcinoma, can be determined by methods known in the art, such as a CT scan, a PET scan, endoscopic ultrasound, sigmoidoscopy, colonoscopy, virtual colonoscopy, and/or DNA stool test. The colorectal cancer can also be diagnosed by obtaining one or more biopsies and evaluating the cells in the biopsy.

Treatment of the cancer is generally initiated after the diagnosis of the cancer, or after the initiation of a precursor condition (such as dysplasia or development of a benign tumor). Treatment can be initiated at the early stages of cancer, for instance, can be initiated before a subject manifests symptoms of a condition, such as during a stage I diagnosis or at the time dysplasia is diagnosed. However, treatment can be initiated during any stage of the disease, such as but not limited to stage I, stage II, stage III and stage IV cancers. In some examples, treatment is administered to these subjects with a benign tumor that can convert into a malignant or even metastatic tumor.

Treatment initiated after the development of a condition, such as malignant cancer, may result in decreasing the severity of the symptoms of one of the conditions, or completely removing the symptoms, or reducing metastasis, tumor volume or number of tumors. In some example, the cancer becomes undetectable following treatment.

In one aspect of the disclosure, the formation of tumors in the treated subject, such as metastasis, is delayed, prevented or decreased. In another aspect, the size of the primary tumor in the treated subject is decreased. In a further aspect, a symptom of the tumor is decreased. In yet another aspect, tumor volume is decreased. Treatment prior to the development of the condition, such as treatment upon detecting dysplasia or an early (benign) precursor condition, is referred to herein as treatment of a subject that is“at risk” of developing the condition. In some embodiments, administration of a

composition can be performed during or after the occurrence of the conditions described herein.

In some examples, treatment using the methods disclosed herein prolongs the time of survival of the subject (e.g., increases survival time by at least 6 months, at least 9 months, at least 12 months, at least 2 years, at least 3 years, or even at least 5 years relative to the absence of the therapy).

The therapeutically effective amount of the LPS and the SCGB3A2 (or SCGB3A2 without LPS) for use in the disclosed methods will depend upon the severity of the disease and the general state of the patient’s health. A therapeutically effective amount of the LPS and SCGB3A2 (or SCGB3A2 without LPS) is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. In one embodiment, a therapeutically effective amount of the LPS and the SCGB3A2 (or SCGB3A2 without LPS) is the amount necessary to inhibit tumor growth (such as growth of a lung tumor), metastasis of the tumor, or the amount that is effective at reducing a sign or a symptom of the tumor. The therapeutically effective amount of the agents administered can vary depending upon the desired effects and the subject to be treated. In some examples, therapeutic amounts are amounts which eliminate or reduce the patient's tumor burden, or which prevent or reduce the growth of metastatic cells, or which prevent or reduce pathological angiogenesis.

The therapeutically effective amount of the LPS and SCGB3A2 (or SCGB3A2 without LPS) can be administered in a single dose, or in several doses, for example daily, during a course of treatment. In one embodiment, a therapeutically effective amount of the LPS and the SCGB3A2 (or SCGB3A2 without LPS) is administered as a single pulse dose, as a bolus dose, or as pulse doses administered over time. In pulse doses, a bolus administration of the LPS and the SCGB3A2 (or SCGB3A2 without LPS) is provided, followed by a time period wherein no LPS or SCGB3A2 is administered to the subject, followed by a second (and optionally subsequent) bolus

administration· In specific, non-limiting examples, pulse doses of the LPS and the SCGB3A2 (or SCGB3A2 without LPS) are administered during the course of a day, or during the course of a week or more.

In some embodiments, the therapeutically effective amount of SCGB3A2 protein can be from about 0.005 mg/kg body weight to about 1 g/kg body weight. In some embodiments, a therapeutically effective amount of the SCGB3A2 protein can be from about 0.1 mg/kg to about 100 mg/kg of body weight. In one specific, non- limiting example, an effective dose is from about 1 mg/kg to about 20 mg/kg, or in even more particular examples, from about 5 mg/kg to about 10 mg/kg of body weight. In some embodiments, a therapeutically effective amount of SCGB3 A2 protein can be from 0.005 to 500 mg/kg of body weight (such as from 0.005 to 0.01 mg/kg, 0.005 to 0.1 mg/kg, 0.005 to 1.0 mg/kg, 0.005 to 10 mg/kg, 0.01 to 0.05 mg/kg, 0.01 to 0.02 mg/kg, 0.01 to 0.1 mg/kg, 0.05 to 0.15 mg/kg, 0.05 to 0.1 mg/kg, 0.1 to 0.15 mg/kg, 0.1 to 0.2 mg/kg, 0.1 to 0.5 mg/kg, 0.1 to 1.0 mg/kg, 0.1 to 1.5 mg/kg, 0.5 to 1.5 mg/kg, 1.0 to 2.0 mg/kg, 1.0 to 5.0 mg/kg, 1.0 to 10.0 kg/kg, or 5.0 to 10.0 mg/kg of body weight).

In some embodiments, the SCGB3A2 administered to the subject can be at a concentration of from about 10 ng/ml to about 1 pg/ml, such as about 10 ng/ml, about 50 ng/ml, about 100 ng/ml, about 200 ng/ml, about 300 ng/ml, about 400 ng/ml, about 500 ng/ml, about 600 ng/ml, about 700 ng/ml, about 800 ng/ml, about 900ng/ml, or about 1 pg/ml.

The LPS administered to the subject can be from any suitable source, such as gram-negative bacteria or synthetically produced. In some embodiments, the LPS is LPS from one of Escherichia coli 011LB4, E. coli K-235, Salmonella typhimurium, or a Ra mutant LPS from E. coli EH-100, E. coli 0111.B8, E. coli 0127:B8, E. coli 0128:B12, E. coli 026.B6, E. coli 055.B5, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica, Salmonella typhosa, Serratia marcesens.

In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 0.005 ng/kg body weight to about 0.5 ng/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 0.01 ng/kg body weight to about 0.5 ng/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 0.1 ng/kg body weight to about 0.5 ng/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3 A2 can be from about 0.2 ng/kg body weight to about 0.5 ng/kg body weight. In some embodiments, the therapeutically effective amount of LPS

administered with SCGB3A2 can be from about 0.3 ng/kg body weight to about 0.5 ng/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 0.4 ng/kg body weight to about 0.5 ng/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 0.2 ng/kg body weight to about 0.4 ng/kg body weight.

In some instances, it is preferred to measure the LPS dosage in endotoxin units. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 0.05 EU/kg body weight to about 5 EU/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 0.1 EU/kg body weight to about 5 EU/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3 A2 can be from about 1 EU /kg body weight to about 5 EU/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 2 EU /kg body weight to about 5 EU/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 3 EU /kg body weight to about 5 EU/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 4 EU /kg body weight to about 5 EU/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 0.05 EU/kg body weight to about 4 EU/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 0.1 EU/kg body weight to about 4 EU/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 1 EU /kg body weight to about 4 EU/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 2 EU /kg body weight to about 4 EU/kg body weight. In some embodiments, the therapeutically effective amount of LPS administered with SCGB3A2 can be from about 3 EU /kg body weight to about 4 EU/kg body weight. In several such embodiments, the therapeutically effective amount of the LPS administered with SCGB3A2 to the subject is less than 5 EU/kg.

In some embodiments, the LPS administered to the subject can be at concentration of from about 10 pg/ml to about 1 pg/ml, such as about 1 pg/ml, about 50 pg/ml, about 100 pg/ml, 500 pg/ml, about 1 ng/ml, about 10 ng/ml, about 50 ng/ml, about 100 ng/ml, about 200 ng/ml, about 300 ng/ml, about 400 ng/ml, about 500 ng/ml, about 600 ng/ml, about 700 ng/ml, about 800 ng/ml, about 900 ng/ml, or about 1 pg/ml.

IV. Compositions and Administration Thereof

The SCGB3A2 protein and LPS (or SCGB3A2 without LPS) can be administered to humans or other animals on whose cells they are effective in various manners such as topically, orally, intravenously, intramuscularly, intraperitoneally, intratumorally, intranasally, intradermally, intrathecally, and subcutaneously, by inhalation, by endotracheal tube, or by injection into the intestine. By way of example, one method of administration to the lungs of an individual is by inhalation through the use of a nebulizer or inhaler. For example, the SCGB3A2 protein and LPS is formulated in an aerosol or particulate and drawn into the lungs using a nebulizer. The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g. the subject, the disease, the disease state involved, and whether the treatment is prophylactic). In cases in which more than one agent or composition is being administered, one or more routes of administration may be used; for example, a chemotherapeutic agent may be administered orally and SCGB3A2 protein and LPS or composition disclosed herein may be administered intravenously. Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.

The SCGB3A2 and LPS (or SCGB3A2 without LPS) administered to the subject are typically included in one or more pharmaceutical compositions (e.g., a single composition including both SCGB3A2 and LPS, or two separate compositions including either SCGB3A2 or LPS) and a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carriers and excipients useful in the disclosed methods are conventional. For instance, parenteral formulations usually comprise fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients that can be included are, for instance, proteins, such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. For instance, in addition to injectable fluids, topical and oral formulations can be employed. Topical preparations can include eye drops, ointments, sprays and the like. Oral formulations can be liquid (e.g. syrups, solutions or suspensions), or solid (e.g. powders, pills, tablets, or capsules). For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

In some embodiments, site-specific administration of the disclosed compounds can be used. Slow-release formulations are known to those of ordinary skill in the art. By way of example, polymers such as bis(p-carboxyphenoxy)propane-sebacic-acid or lecithin suspensions may be used to provide sustained intra-tumoral release.

The formulations can be prepared by combining a SCGB3A2 protein and LPS (or

SCGB3A2 without LPS) uniformly and intimately with liquid carriers or finely divided solid carriers or both. The formulations can also be prepared by combining microparticles including or consisting of the nanoparticles uniformly and intimately with liquid carriers or finely divided solid carriers or both.

In some embodiments, the pharmaceutical composition comprising SCGB3A2 protein and LPS comprises from about 1 pg/ml to about 1 mg/ml SCGB3A2 protein, and from more than 0.01 EU/ml to about 5 EU/ml LPS.

The pharmaceutical compositions that comprise SCGB3A2 protein and LPS (or SCGB3A2 without LPS) can be formulated in unit dosage form, suitable for individual administration of precise dosages. The amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated. Multiple treatments are envisioned, such as over defined intervals of time, such as daily, bi-weekly, weekly, bi-monthly or monthly, such that chronic administration is achieved. Administration may begin whenever appropriate as determined by the treating physician.

The compositions or pharmaceutical compositions can include a nanoparticle including SCGB3A2 protein and LPS (or SCGB3A2 without LPS), which can be administered locally, such as by pulmonary inhalation or intra-tracheal delivery. When nanoparticles are provided, or microparticles including or consisting of these nanoparticles are provided, e.g. for inhalation or infusion, they are generally suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate- phosphoric acid, and sodium acetate-acetic acid buffers. A form of repository or“depot” slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery.

Lor administration by inhalation, nanoparticles or microparticles including the SCGB3A2 protein and LPS (or SCGB3A2 without LPS) can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The site of particle deposition within the respiratory tract is demarcated based on particle size. In one example, particles of about 1 to about 500 microns are utilized, such as particles of about 25 to about 250 microns, or about 10 to about 25 microns are utilized. In other embodiments, particles of about 1 to 50 microns are utilized. For use in a metered dose inhaler, for administration to lungs particles of less than about 10 microns, such as particles of about 2 to about 8 microns, such as about 1 to about 5 microns, such as particles of 2 to 3 microns, can be utilized.

Methods of administration include injection for which the SCGB3A2 protein or

compositions including these compounds are provided in a nontoxic pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oils, ethyl oleate, or liposomes.

Site-specific administration of the disclosed compounds can be used, for instance by applying LPS and SCGB3A2 protein to a pre-cancerous region, a region of tissue from which a neoplasm has been removed, or a region suspected of being prone to neoplastic development. In some embodiments, sustained intra-tumoral (or near-tumoral) release of the pharmaceutical preparation that comprises a therapeutically effective amount of SCGB3A2 protein and LPS may be beneficial.

The pharmaceutical compositions that comprise a SCGB3A2 nucleic acid and/or protein and LPS can be formulated in unit dosage form, suitable for individual administration of precise dosages. For example, one possible unit dosage can contain from about 1 ng to about 1 pg of SCGB3A2 protein and from about 1 pg to 1 pg of LPS. The amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated

In some embodiments, the SCGB3A2 protein is administered to the subject by

administration of a nucleic acid molecule (such as plasmid DNA or a viral vector) encoding the SCGB3A2 protein, to provide in vivo SCGB3A2 protein production using the cellular machinery of the subject. Administration by nucleic acid constructs is well known in the art and taught, for example, in U.S. Patent No. 5,643,578, and U.S. Patent No. 5,593,972 and U.S. Patent No.

5,817,637. U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding a particular protein to an organism. The methods include liposomal delivery of the nucleic acids. Liposomes can further enclose LPS. Such methods can be applied to the production of a protein of interest in the subject by one of ordinary skill in the art. One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the SCGB3A2 protein can be placed under the control of a promoter to increase expression.

In another approach to using nucleic acids, SCGB3A2 protein can also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirus or other viral vectors can be used to express the SCGB3A2 protein.

In one embodiment, a nucleic acid encoding SCGB3A2 protein is introduced directly into cells. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS™ Gene Gun. The nucleic acids can be“naked,” consisting of plasmids under control of a strong promoter.

Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 pg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466).

In some embodiments, an additional anti-cancer agent can be administered to the subject along with the SCGB3A2 and LPS to treat the cancer in the subject. Thus, additional agents can be administered to the subject, such as a cytokine, a chemokine, or a chemotherapeutic agent. These can be included in the disclosed pharmaceutical compositions. A cytokine can be administered, such as interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), or interferon, such as interferon (IFN) b. In one example, for the prevention and treatment of cancer, surgical treatment can be administered to the subject. In one example, this administration is sequential. In other examples, this administration is simultaneous.

Examples of chemotherapeutic agents are alkylating agents, antimetabolites, natural products, or hormones and their antagonists. Examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or

chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine). Examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine. Examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (such as L-asparaginase). Examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide). Examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as

hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU,

Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP- 16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-l l), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol. Non-limiting examples of immun om odul ators that can be used include AS-101 (Wyeth- Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

The subject can be administered an immunotherapy. In some embodiments, the subject is administered a PD-l antagonist, such as antibody that specifically binds PD-l or PD-L1, such as MPDL3280A. In other embodiments, the subject is administered ERBITUX® (cetuximab).

Without further elaboration, it is believed that one skilled in the art can, using this description, utilize the present discoveries to their fullest extent. The following examples are illustrative only, and not limiting of the disclosure in any way whatsoever.

EXAMPLE

SCGB3A2~I.PS complex u take leads to pyroptotic death of cancer cells

This example illustrates that SCGB3A2 is an LPS binding protein and that the SCGB3A2- LPS complex binds to cancer cells to induce pyroptotic cell death via the non-canonical inflammasome pathway and pyroptotic death.

SCGB3A2 inhibits LLC cell growth in vitro and in vivo

To determine whether SCGB3A2 has any influence on cancer cell growth, a CCK8 (cell counting kit 8) assay was performed using murine Lewis lung carcinoma (LLC) cells. The proliferation of LLC cells was markedly suppressed by mouse recombinant SCGB3A2 (FIG. 1A). This in vitro effect of SCGB3A2 was also observed in vivo in the LLC cells intravenous metastasis model using wild-type C57BL/6 mice in conjunction with administration of SCGB3A2 (FIGS. 1B- 1E). To confirm the tumor growth inhibition roles of SCGB3A2 in vivo, Scgb3a2- null mice were subjected to the metastasis model described in Kido et al, Mediators Tnflamm 2014, 216465, 2014. Mice null for Scgb3a2 developed far greater numbers of lung surface tumors than wild-type littermates when LLC cells were intravenously injected (FIG. 1F). Furthermore, administration of recombinant mouse SCGB3A2 to Scgb3a2- null mice clearly rescued the Scgb3a2- null phenotypes of LLC cell lung metastasis (FIGS. 1G-1I). These results indicate the importance of SCGB3A2 in the suppression of LLC cell tumor development in lungs in vivo.

SCGB3A2 binds to LPS

For the above experiments, several preparations of recombinant SCGB3A2 (mouse and human) were used from various sources as described in Methods. However, an unexpected phenomenon was observed where some sources of recombinant SCGB3A2 had almost no effect on LLC cell growth inhibition (FIG. 8A). This phenomenon was found to be associated with the level of endotoxin (LPS) contained in the various preparations (Table 1).

Table 1. LPS contents in various preparations/batches of recombinant SCGB3A2

Moreover, it was realized that whenever the endotoxin was removed from the preparation, the final SCGB3A2 protein yield was drastically reduced. Because SCGB3A2 is abundantly expressed in airway epithelial cells which are exposed to various microorganisms and LPS derived from bacteria, it was postulated that SCGB3A2 may bind to and sequester LPS. Further, while inflammation is thought to be coupled with cancer metastasis, paradoxically endotoxins or ensuing enhanced immunity may inhibit some cancer growth. Indeed, the growth of LLC cells was strongly inhibited by small amounts of LPS (FIG. 2A). To test if SCGB3A2 interacts with LPS, imidazole and zinc salt staining was performed according to previously described methods (FIGS. 2B and 2C) (Rodriguez and Hardy, Anal Biochem 485, 72-80, 2015). Crude LPS (Oll l:B4) barely migrated into the gel and remained near the well, due to high-molecular mass aggregation (FIG. 2B, lane 1). Pre-incubation with SCGB3A2 (human SCGB3A2 (Cl); see Table 1; unless otherwise noted, this lot was mainly used in the studies in this example) produced a broad diffuse band in dose dependent manner, indicating that SCGB3A2 interacted with and dramatically enhanced the electrophoretic mobility of LPS (FIG. 2B, lane 2- 5, and FIG. 2C). Rough A form (Ra-LPS) and other serotypes of LPS produced the same results (FIG. 8B). To further confirm that SCGB3A2 directly binds to LPS, streptavidin pull-down assays were performed using LPS-Biotin and recombinant SCGB3A2 (FIG. 2D). The results clearly showed that SCGB3A2 is an LPS binding protein. The ability of SCGB3A2 to disaggregate LPS micelles was further demonstrated by the dynamic light scattering (DLS) method (FIG. 2E and FIGS. 8C-8E). Thus, SCGB3A2 is an LPS binding protein and has powerful LPS dissociation properties, against both smooth and rough forms of LPS.

To determine if LPS alone is sufficient or the combination of LPS+SCGB3A2 is required for the inhibition of growth and metastasis of LLC cells in vivo, LLC cell intravenous metastasis xenograft experiments were carried out, in which various amounts of LPS, estimated in the recombinant protein SCGB3A2 preparations, were administered for seven consecutive days in the first week after LLC cells injection (see FIG. IB). The number of tumors obtained was compared with that obtained with administration of recombinant SCGB3A2 without exogenously added LPS. Human SCGB3A2(C1) alone showed drastic inhibition of LLC cells growth, while the amount of LPS contained in the recombinant SCGB3A2 preparation Cl or C3, or high concentration did not show any statistically significant differences in tumor numbers compared with PBS administration (FIG. 2F). Moreover, LPS-treated lungs showed much larger lesions than did SCGB3A2-treated lung tumors, which sometimes encompassed the entire lobes, demonstrating the fundamental differences between LPS alone and SCGB3A2 administration (FIG. 2G).

SDC1 is a receptor for SCGB3A2

A receptor for SCGB3A2 involved in the SCGB3A2 signaling was unbiasedly identified using human protein microarray analysis (FIG. 3A). Among the top 116 proteins, 13 proteins were selected as possible candidates for the SCGB3A2 receptor as a cell surface protein (FIG. 3A and Table 2). Table 2. SCGB3A2 candidate receptors identified by human protein interaction array

To confirm a direct interaction with SCGB3A2, pulldown assays were performed, in which syndecan-l (SDC1) and podoplanin (PDPN, Tl-alpha) showed positive interaction with SCGB3A2 (FIG. 3B). PDPN is known as a marker for alveolar type I epithelial cells in lung, while SDC1 was moderately expressed in proximal airway epithelial cells (FIG. 3C), suggesting a possible relationship between SCGB3A2 and SDC1 for lung airway homeostasis. Therefore, this study focused on SDC1.

SDC1 was found to be highly expressed on LLC cells surfaces in vitro as well as in metastatic LLC cells in vivo (FIGS. 3D and 9A). In contrast, the B16F10 mouse melanoma cell line, which exhibited less SCGB3A2-dependent growth suppression effects than LLC in vitro, showed focal expression of SDC1 near cell nuclei and faint staining at cell-to-cell contact sites (FIG. 3D), while the total cell surface staining was low compared to LLC cells. Further analyses supported the robust expression of SDC1 on the surface of LLC cells (FIGS. 3E and 9B), and their binding to SCGB3A2 (FIG. 3F). LLC cells stably expressing shRNA-SDCl (LLC-sh-SDCl, FIG. 9C) showed diminished SCGB3A2 binding (FIG. 3G). In addition, ARH-77 human myeloma cell line, which lacks detectable SDC1 (Ridley et al, 1993), and ARH-77 cells over-expressing mouse SDC1 (ARH-77-mSDCl (Dhodapkar et al, 1998; Liebersbach and Sanderson, 1994), see FIG. 9D) verified the SCGB3A2-SDC1 binding interaction. ARH-77-mSDCl enhanced SCGB3A2 binding compared to the parental cells (FIG. 9E). Syndecans are a family of transmembrane heparan sulfate proteoglycan (HSPG). To determine the domain of SDC1 that interacts with SCGB3A2, heparin was used to inhibit the function of heparan sulfate chains (HS). Heparin addition significantly inhibited the binding of SCGB3A2 to both LLC (FIG. 3H) and ARH-77-mSDCl cells (FIG. 9F), suggesting that HS on SDC1 may play a role in SCGB3A2 binding.

SCGB3A2 accumulates on the uropod and is incorporated through clathrin-mediated endocytosis in LLC cells

To reveal the precise binding site of SCGB3A2 on LLC cell surfaces, immunofluorescence analysis was performed using anti-SCGB3A2 and anti-SDCl ectodomain antibodies. ARH-77- mSDCl cells were also used in this analysis (FIG. 4). Without stimulation with SCGB3A2, both LLC and ARH-77-mSDCl cells had evenly distributed SDC1 on the cell surface (FIG. 4A). After stimulation with SCGB3A2, the SDC1 signal became relatively concentrated on cell protrusions equivalent to the uropod structure of myeloma, which co-localized with SCGB3A2 (FIG. 4A). Staining of ICAM-l, a uropod marker, confirmed co-localization of SDC1 and SCGB3A2 on the uropods of both LLC and ARH-77-mSDCl cells. Interestingly, when LLC cells were incubated for a short time with LPS and SCGB3A2, Alexa-labeled LPS (LPSA488, FIG. 10A), SCGB3A2, and clathrin, a key protein for endocytosis, all co-localized in uropod (FIG. 4B). This pattern of clathrin localization was similar to those previously reported using T lymphocyte. Upon further incubation, LLC cells appeared to have incorporated SCGB3A2 into the cells as visualized using a HaloTag (HT)-SCGB3A2 fusion protein (FIGS. 10B and 10C). Clathrin expression was localized near the incorporated SCGB3A2 signals (FIG. 10C), suggesting that the LPS-SCGB3A2 complex enters LLC cells via binding to SDC1 followed by clathrin-dependent endocytosis. Further, live cell imaging clearly showed that SCGB3A2-HT was incorporated into LLC-sh-Control cells after overnight incubation, while very low signals were observed in LLCsh-SDCl cells (FIG. 10D). Computer modeling analysis provided further evidence that SCGB3A2 binds to both LPS and SDC1 when it forms a tetramer (FIG. 4C and FIGS. 11A-110). In fact, SCGB3A2 tends to form oligomers in vitro, demonstrating the validity of the computer modeling (FIG. 110, see FIG. 2C CBB staining, also Cai et al. , Am J Physiol Lung Cell Mol Physiol 306, L10-22, 2014; and Niimi et al, Mol Endocrinol 15, 2021-2036, 2001).

SCGB3A2 functions as a chaperone to deliver LPS into the cytosol and activates caspase- 11/NLRP3 inflammasome foci formation

Intracellular LPS has been demonstrated to trigger caspase-4/ll activation and the non- canonical inflammasome pathway (see Hagar et al, Science 341, 1250-1253, 2013; and Kayagaki et al, Science 341, 1246-1249, 2013). To address the possibility that SCGB3A2 enhances TLR4 priming canonical signals via SDC1 binding, transferring LPS to TLR4, LLC cells stably expressing sh-TLR4 (LLC-sh-TLR4) were established (FIG. 12A), and SCGB3A2 binding and uptake were compared with those of LLCsh-Control and LLC-sh-SDCl cells (FIGS. 12B and 12C). SCGB3A2 enhanced binding of LPS to LLC-sh-TLR4 cells at similar level to that of LLC-sh- Control, while LLC-sh-SDCl cells showed little binding of LPS (FIG. 12B). In addition,

SCGB3A2 and LPS were incorporated into LLC-sh-TLR4 cells and appeared to co-localize within the cytosol (FIG. 12C). These data, together with data in FIG. 10D, suggest that SCGB3A2 is important for LPS uptake and that SDC1, not TLR4, is required for SCGB3A2-LPS incorporation.

To confirm the cytosolic localization of LPS, LLC cells were visualized with Alexa-labeled LPS (LPS_A594), anti-EEAl (early endosomes marker) and anti-Lampl (lysosomal marker) antibodies (FIGS. 4D and 4E). At an early time point, most of the LPS staining was co-localized with EEA1, which was clearly enhanced when cells were co-incubated with SCGB3A2 (FIG. 4D). At a later time point, however, some of the LPS staining did not overlap with either EEA1 or LAMP1 (FIG. 4E). With SDC1 staining depicting the plasma membrane, LPS staining was clearly visible inside the membrane, which differed from the EEA1 distribution pattern (FIG. 4F). These results indicate that LPS is transported into the cytosol of LLC cells through an SCGB3A2- dependent mechanism.

Next whether LPS transport into cytosol of LLC cells triggers non-canonical inflammasome pathway was examined using LLC-sh-TLR4 cells. This was because TLR4 signaling also enhances pro-caspase-l l/NLRP3 expression via the canonical inflammasome pathway (Kayagaki et al, Science 341, 1246-1249, 2013). SCGB3A2 + LPS increased pro-caspase-l l and NLRP3, while caspase-l expression, another caspase involved in inflammation was not significantly different (FIG. 4G). Caspase-ll processing and IL-Ib expression/processing were not detected at the protein level in LLC-sh-TLR4 and LLC-sh-Control cells.

The importance of sensing LPS and triggering caspase-l l and NLRP3 activation in host defense has been mainly studied using macrophage. Moreover, macrophage are key players both for lung homeostasis and the tumor microenvironment. Hence, the effect of SCGB3A2 on mouse macrophage-like RAW264.7 cells, which express SDC1 on the cell surface (FIG. 13 A), was next examined. Co-incubation of RAW264.7 cells with SCGB3A2+LPS clearly enhanced expression of caspase-ll and NLRP3, followed by IL-Ib up-regulation and maturation, while SCGB3A2 or LPS alone did not (FIG. 4H). Heparin co-incubation abrogated caspase-l l/NLRP3/IL-l expression by SCGB3A2+LPS. SCGB3A2 enhanced IL-Ib secretion from RAW264.7 cells without priming signals, which was inhibited by the addition of heparin (FIG. 13B). A lactate dehydrogenase (LDH) cytotoxicity assay showed that RAW264.7 cells exhibited greater cytotoxicity by

SCGB3A2+LPS compared to the individual treatments (FIG. 13C).

In LLC cells under SCGB3A2+LPS, caspase-ll expression was upregulated in a diffused distribution pattern in the entire area and showed specific foci (FIG. 41). Importantly, the caspase- 11 foci overlapped with incorporated LPS (FIG. 41). The expression of NLRP3 was also clearly up- regulated by LPS+SCGB3A2 and accumulated around the caspase-ll foci. When LPS was introduced into LLC cells using a DNA transfection reagent, LLC cells showed increased intracellular LPS signals and caspase-l l foci, overlapped with LPS (FIG. 4J), confirming that the formation of caspase-ll foci is mediated by LPS introduction into the cytosol of LLC cells.

Caspase-ll foci formation and NLRP3 upregulation driven by LPS+SCGB3A2 were also observed in RAW264.7 cells (FIG. 13D). These results confirm that SCGB3A2 facilitates the delivery of LPS into the cytosol, in concert with the enhancement of non-canonical inflammasome signaling.

To confirm the importance of clathrin-mediated endocytosis of LPS via the SCGB3A2- SDC1 pathway for killing of LLC cells, the effect of ciathrin inhibitor, Dynasore on the growth of LLC cells was examined in vitro (FIGS. 4K-4M). LLC cells had strong focal staining of

SCGB3A2-HT and LPS_A44S at the corresponding locations to each other, while Dynasore potently inhibited the incorporation of SCGB3A2 and LPS into the cytosol of LLC cells (FIG. 4K) and abrogated the activation of caspase-ll (FIG. 4L). LDH release from LLC cells as indication for cytotoxicity was slightly upregulated by LPS +SCGB3A2, while this upregulation was not observed when cells were treated with either Dynasore or Wedelolactone (caspase-11 inhibitor) (Kobori et at, Ceil Death Differ 11: 123-130, 2004) (FIGS. 4M and 40). Furthermore, when LLC- sh-casp-11 cells (FIG. 4P) were subjected to the intravenous metastasis model with or without SCGB3A2 administration, they did not show any significantly different numbers of lung tumors after SCGB3A2 administration, in sharp contrast to the results with control LLC cells (FIG. 4N). These results clearly indicate the importance of clathrin-mediated endocytosis of LPS +SCGB3A2 and caspase-l l activation for the killing of LLC cells in vivo.

SCGB3A2-LPS promotes pytoptotic cell death of LLC cells

The SCGB3A2+LPS complex promoted pyroptotic cell death morphology in cultured LLC cells (membrane swelling; FIG. 5A). CCK8 assay confirmed the upregulation of pyroptotic cell death of LLC cells by essentially endotoxin-free SCGB3 A2 plus a small amount of LPS (FIG. 5B). Furthermore, flow cytometry analysis revealed the upregulation of propidium iodide (PI) positive cell death by SCGB3A2+LPS (FIG. 5C), demonstrating the formation of cell membrane pores, the characteristic feature of pyroptosis, induced by SCGB3A2+LPS. Next, the induction of cell death by SCGB3A2 in vivo was examined in the LLC cell intravenous metastasis model (FIG. 5D). Large necrotic areas were found in lung tumors from mice treated with early intravenous administration of SCGB3A2 (lst and 2nd week) (FIG. 5D). Importantly, these necrotic areas showed enhanced expression of both caspase-ll and NLRP3, demonstrating that the pyroptotic cell death occurred through caspase-l l and NLRP3 activation (FIGS. 5E and 5F). These results clearly indicate that SCGB3A2 significantly promotes pyroptotic death of LLC cells both in vivo and in vitro.

LLC-sh-SDCl cells attenuate SCGB3A2-mediated inhibition of metastasis

in the mouse LLC model

LLC-sh-SDCl cells showed reduced susceptibility to the cytotoxic effects of

LPS+SCGB3A2 complex in vitro (FIG. 6A), accompanied by minimal enhancement of caspase-ll foci formation by LPS+SCGB3A2 (FIG. 6B, see FIG. 41). Heparin addition abrogated the increase of caspase-ll foci in LLC-sh-Control cells (FIG. 6B), confirming the crucial role of heparin sulfate and SDC1 for caspase-ll foci formation. In vivo sensitivity of LLC-sh-SDCl cells to SCGB3A2- mediated inhibition of metastasis was next analyzed. Tumor numbers in mice that received LLC12 sh-SDCl cells and SCGB3A2 were not significantly different from those that received LLC-sh- SDCl cells and PBS, while tumor numbers with LLC-sh-Control cells were significantly reduced by SCGB3A2 co-injection, similar to that observed in FIG. 1 (FIGS. 6C and 6D). These experiments confirmed a role for SDC1 in SCGB3A2-mediated inhibition of LLC cell growth and metastasis in vivo. Next, to understand the reason for the differences in response to SCGB3A2 between LLC (susceptible) and B16F10 (resistant) cells, the baseline mRNA expression from inflammasome-related genes were examined (FIG. 6E). As a result, CaspH, Nlrp3, Aim2, Gsdmd, and lllb mRNAs were highly expressed only in LLC cells, suggesting that LLC cells have the machinery to activate a non-canonical inflammasome pathway driven by caspase- 11 in combination with higher expression levels of cell surface SDC1 (see FIGS. 3D and 3E). Lastly, the effect of SCGB3A2 on the survival of lung-specific Kras^G12D mutant mice was examined using Kras^G12D ;Scgb3a2(fl/fl) and the littermate Kras^G12D ;Scgb3a2(fl/+ } mice (FIG. 6F). Due to lung-specific activation of the Kras^Gl2D allele, these mice developed lung cancer within 4 months of age. Kra$^Gl2D;Scgb3a2(fl/fl) mice clearly showed a poorer survival rate

than Kras^G12D;Scgb3a2(fl/+ ) mice.

Differential SDC1 and caspase-4 expression patterns determine the effect of SCGB3A2 in human cancer cell lines.

To examine if the differences found in SDCl/caspase-l l expression patterns between LLC and B16F10 mouse cancer cells that determines their susceptibility to SCGB3A2 are also found in human cancer cells, a panel of 20 human cancer-derived cells (17 lung, 2 colon, and 1 cervical cancers) were analyzed for the expression of mRNA encoding SDC1 and caspase-4 ( CASP4 , human equivalent of caspase-l l in mice) (FIGS. 7A-C, 14 and 15, Table 3), and CCK8 assays to evaluate the effect of SCGB3A2+LPS on the growth of these cancer cells (FIG. 7E, Table 3). Interestingly, a high positive correlation was obtained between mRNA expression levels of SDC1 and CASP4, suggesting an interaction at genomic and/or genetic levels between the SDC1 receptor and caspase-4 (FIGS. 7A-7D). Among the 20 cell lines, 7 cancer cells (5 non-small cell lung cancers (NSCLCs); NCI-H596, H358, H322, A549, and H157, and 2 colorectal cancers; HCT116 and SW620) showed approximately 20% growth reduction by SCGB3A2+LPS treatment (FIGS.

7C and 7E). In contrast, none of small cell lung cancers (SCLCs) analyzed (NCI-H526, H417, H146, H446, H82, and H1688) exhibited obvious response to SCGB3A2 (FIG. 7E for H82, Table 3). To further understand the underlying mechanism that determines the differences in response to SCGB3A2+LPS, the expression patterns of SDC1 receptor protein and HS were evaluated by immunofluorescence analysis (FIGS. 7F and 14, Table 3), and flow cytometry analysis for their cell surface expression levels (FIGS. 7G, 7H, and 15, Table 3). Immunofluorescence analysis revealed that all of 7 SCGB3A2-susceptible cancer cells expressed SDC1 on their cell surfaces (FIGS. 7F, H596, H358, SW620 and H157 white arrowheads, and FIG. 14 for H322 (14D), A549 (14F) and HCT116 (14K) white arrowheads). In contrast, non-susceptible cells showed little SDC1 expressions (FIG. 7F, H82 and FIG. 14, H417 (141), H146 (14L) and H526 (14R)) or showed cytoplasmic/nucleus/perinuclear SDC1 expression (FIG. 7F, H292, white arrowhead and FIG. 14, H1688 (14G), H1703 (14J), H446 (14M) and H727 (14Q)). Interestingly, H1155 cells showed membranous SDC1 expression by both immunofluorescence and flow cytometric analysis (FIGS. 7F and 7G), while they did not express detectable levels of HS (FIG. 7H), and they did not respond to SCGB3A2+LPS (FIG. 7E). Further, H1299 cells showed very little SDC1 but abundant HS expression, while they were not susceptible to SCGB3A2+LPS (FIG. 14S and 15S, Table 3).

These results suggest the importance of specific HS sequence of SDC1 for the SCGB3A2 signaling. Collectively, these results suggest that the synergistic high level expression of both SDC1 with specific HS sequence and caspase-4 is critical for SCGB3A2+LPS effects on the growth of cancer cells. Based on these results, a new model for SCGB3A2 delivery of LPS and activation of caspase- 11 (caspase-4) pathway via SDC1 receptor signaling, leading to pyroptosis of cancer cells, is proposed (FIG. 71).

Table 3. Summary of SDC1, HS and CASP4 expressions in human cell lines and their susceptibilities to SCGB3A2.

In Table 3, the numbers for qPCR are relative mRNA levels compared with the value of A549 cell, set as 1. FACS mean indicates a mean of subtraction of stained cells and unstained control. FACS median means a median of subtraction of stained cells and unstained control. For both calculations, representative graphs from 2-5 independent experiments were used.

DISCUSSION

SCGB3A2 is a member of the secretoglobin family of proteins, which share a common four helical bundle subunit structure, exist as dimers, tetramers, and other oligomers, and some of which have also been implicated in tumor suppression (Mukherjee et al, Endocr Rev 28, 707-725, 2007) without a clear understanding yet of the mechanistic pathway(s). The data presented in this example elucidate and describe a new pathway impacted by SCGB3A2 functioning as a tumor suppressor protein. Previously it was demonstrated that SCGB3A2 functions as an anti inflammatory and anti-fibrotic agent in the lung (Cai and Kimura, PLoS One 10, e0l42497, 2015; Cai et al, Am J Physiol Lung Cell Mol Physiol 306, L10-22, 2014; Chiba et al, Am J Respir Crit Care Med 173, 958-964, 2006; Kido et al., Mediators Tnflamm 2014, 216465, 2014; Kurotani et al., J Biol Chem 286, 19682-19692, 2011; and Yoneda et al., Int Arch Allergy Immunol 171, 36-44, 2016). Because SCGB3A2 is mainly secreted by club cells in lung airways, it is reasonable to assume that a primary function of SCGB3A2 is to protect the hosts from pathogens and pathogen- associated molecular patterns such as LPS. This example shows that SCGB3A2 binds to and facilitates delivery of LPS into the cytosolic compartment through specific binding with SDC1, resulting in cell death via an inflammatory pathway leading to pyroptosis. This is commonly seen in the macrophage cell line RAW264.7, suggesting a possible conserved role for SCGB3A2 in host defense and enhancing the immune response through the non-canonical inflammasome pathway of pyroptosis. Notably, in the case of LLC cells, the uptake of SCGB3 A2- sequestered small amount of LPS triggers inflammatory cell death, probably because of the abundant SDC1 expression on their cell surface. It is noteworthy that caspase-l l and human caspase-4/5 are specific to mammals (Kayagaki et al, Science 341, 1246-1249, 2015), while the SCGB superfamily of proteins, including SCGB3A2, have also evolved in mammalian lineages (Jackson et al, Hum Genomics 5, 691-702, 2011), suggesting the co-emergent evolution as an“input-output” for defense from invading pathogens.

SDC1 localization to uropods is functionally important as uropods accumulate growth factors and connect them at cell-to-cell contact points or junctions (Borset et al. Blood 96, 2528- 2536, 2000; Yang et al, J Biol Chem 278, 12888-12893, 2003). It has been demonstrated that the SDCl-specific HS sequence is important for targeting SDC1 to uropods (Borset et al. Blood 96, 2528-2536, 2000). The results presented herein that heparin treatment dramatically reduces SCGB3A2 and LPS binding and their incorporation into cells, as well as caspase-l l and NLRP3 induction, and SCGB3A2 has little effect against both NCI-H1155 which expresses SDC1 but lacks HS, and NCI-H1299 which expresses little SDC1 but abundant HS, suggest that SCGB3A2 appears to interact with the HS moiety of SDC1, which is concentrated in the membrane uropods.

It was reported that the non-canonical inflammasome pathway governed by caspase- 4/caspase-l l, intrinsic to intestinal epithelial cells, plays a critical role in antimicrobial defense, causing pyroptotic cell death and shedding of infected cells (Knodler et al., Cell Host Microbe 16, 249-256, 2014). These events could limit pathogen colonization of the intestinal epithelium.

Likewise, it’s conceivable that lung airway epithelial cells have an intrinsic non-canonical inflammasome pathway for antimicrobial defense, through the SCGB3A2 and SDC1 interaction. Moreover, the present results suggest that this non-canonical inflammasome pathway is retained in some cancer cells and this property could be used for cancer treatment. Importantly, it was reported that newborn Sdcl (-/-) mouse lungs show marked resistance against P. aeruginosa infection (Park et al., 2001). This study was extended to show the biological function of SDC1 in lung epithelial cells from a simple cell membrane receptor for growth factors and chemokines to that of modulating microbial pathogenesis and host defense (Park et al., 2001).

The role of SCGB3A2 as a chaperon to deliver LPS to cell cytosols may initially be established to protect host cells from infection, while this mechanism may have evolved to protect host from cancer development by activation of the non-canonical inflammasome signaling pathway. Anti-tumor effects of endotoxin/LPS has been known for decades while the effects are still controversial; one reason is because the effects vary depending on different cancers (Lundin and Checkoway, 2009; Ribi et al. , 1983). Our results could provide the reason for the various sensitivities of different cancer cells to endotoxin. In this report, we analyzed the SDC1/HS expression and the non-canonical inflammasome CASP4 expression in various human cancer cells and evaluated their susceptibilities to SCGB3A2 and LPS. Cancer cells with high expression of membranous SDC1/HS and CASP4 are highly sensitive to SCGB3A2 as compared with cells with low level SDC1/HS and CASP4 expression. In the current study, 5 NSCLC and 2 colon cancer cells were found to be susceptible to SCGB3A2+LPS treatment. The lung and colon are the organs which are constantly exposed to bacteria. Their machinery to fight against microorganism infection triggering the non-canonical inflammasome pathway might have been retained in these cancer cells. In contrast, none of SCLCs analyzed exhibited high levels of membrane SDC1 expression, and they were refractory to SCGB3A2+LPS. SCLCs originate from neuroendocrine, but not epithelial cells, and are the more aggressive type of lung cancers as compared with NSCLCs.

Experimental Procedures

Antibodies and reagents. Recombinant SCGB3A2 proteins were obtained from the following sources: mouse SCGB3A2 from the NIH Core Facility (Kurotani et al, Am J Respir Crit Care Med, 178, 389-398, 2008), Holzel Diagnostics (Germany), and CosmoBio USA; human SCGB3A2 from Therabron Therapeutics (Cai et al, Am J Physiol Lung Cell Mol Physiol 306:Ll0- 22, 2014). Unless indicated otherwise, human SCGB3A2 from Therabron Therapeutics was used in the experiments described herein. Anti-mouse SCGB3A2 antibody was obtained from rabbit as previously described (Niimi et al, Mol Endocrinol 15, 2021-2036, 2001), and anti-human

SCGB3A2 antibody was from Therabron Therapeutics. Anti-mouse and anti-human SDC1 ectodomain antibodies were provided by Dr. Pyong W Park (Harvard Medical School). PE-rat anti mouse SDC1 antibody (clone 281.2) was from BD Pharmingen; anti-Clathrin heavy chain (P1663), anti-EEAl (C45B10), anti- 1 L- 1 b (clone 3A6) were purchased from Cell Signaling Technology; anti-Caspase- 11 (clone 17D9), anti-ICAM-l (MA5407), anti-Caspase-l (clone 5B10) were from Thermo Fisher Scientific; anti-NLRP3 (AG-20B-0014) was from Adipogen; anti-HS antibody (clone F58-10E4) was from Amsbio; anti-LAMPl(sc- 17768) from Santa Cruz; anti-HaloTag antibody (G9218) was from Promega; anti-GAPDH monoclonal antibody (60004-l-lg) was from Proteintech; anti-LPS antibody (ab35654) was from Abeam. LPS from Escherichia coli 011 UB4 (E4391), Ra mutant LPS from E. coli EH-100 (L9641), LPS from E. coli K-235 (L2018), LPS from Salmonella typhimurium (L2262), heparin sodium salt from porcine intestinal mucosa (H3393), imidazole (15513), and zinc sulfate solution (Z2876) were all purchased from Sigma- Aldrich. Coomassie Brilliant Blue R-250 (20278) was from Thermo Fisher Scientific. Dynasore (A122726) and Wedelolactone (A14804) were from AdooQ Bioscien. LPS-EB Biotin (tlrl-3blps) was from InvivoGen.

Cell culture. The LLC cells used in this study were the LLC-Mhi cell line (obtained from Dr. Glenn Merlino, NCI), which is a high metastatic sub-line derived from LLC tumors described previously (Day et al, Int J Cancer 130, 190-199, 2012). Human lung cancer-derived cell lines, A549, NCI-H322, H358, H1299, H146, H596, H82, H526, H417, H446, H727, H292, H1155, H157, H1688 cells were obtained from Dr. Curt Harris (NCI), SW620 colon cancer derived cell line was from the DTP (Developmental Therapeutics Program, NCI/DCTD (Division of Cancer Treatments and Diagnostics)) tumor cell lines repository. B16F10, RAW264.7, HEK293, COS-l, NCI-H838, H1703 and HeLa cells were purchased from American Type Culture Collection (ATCC). HCT116 was provided from Dr. Frank Gonzalez (NCI). ARH-77 and ARH-77-mSDCl cells were provided by Dr. Ralph D. Sanderson (University of Alabama at Birmingham). LLC, B16F10 cells and most of human cell lines were cultured in RPMI 1640 Medium (LONZA), all with or without heat-inactivated fetal bovine serum (FBS), supplemented with

penicillin/streptomycin (1:100) at 37°C, 5% C02. Culture of LLC cells was carried out under various concentrations of FBS, as indicated in the Figure legends. HCT116 cells were cultured in McCoy's 5A Medium (LONZA). For LPS stimulation, RAW264.7 cells were cultured in OPTI- MEM™ I reduced serum medium (Thermo Fisher Scientific) for times indicated in the text. LPS transfection was performed using X-tremeGene™ HP DNA transfection reagent (Roche Applied Science).

Protein microarray. SCGB3A2 binding proteins were identified using Protoarray™ Human Protein Microarray v5.0 Protein- Protein Interaction Kit for biotinylated proteins (Thermo Fisher Scientific, PAH0525101, >9,000 proteins included). Experiments were carried out according to procedures provided by the manufacturer. First, a biotin label was introduced into recombinant human SCGB3A2 protein using Biotin-XX Microscale Protein Labeling Kit (Thermo Fisher Scientific B30010), which was then used to probe Protoarray Human protein microarrays. The microarrays were washed with washing buffer (PBS containing 10% Synthetic Block (included in the kit) and 0.1% Tween 20 (Thermo Fisher Scientific)), and probed with Alexa Fluor 647 conjugated streptavidin (included in the kit). After washing, the microarrays were dried and scanned by a fluorescent microarray scanner (Perkin Elmer, Scanarray Express) to obtain the data. Software for the data analysis (Protoarray Prospector) was also provided by the manufacturer.

RNA interference by retrovirus based shRNA. The shRNA constructs were purchased from transOMIC for mouse SDC1, from ORIGENE for mouse TLR4. Retroviral constructs were transfected into Phoenix packaging cells by using XtremeGene™ HP DNA transfection reagent (Roche Applied Science). Drug selection and cell cloning were conducted in the presence of 2 pg/ml puromycin by the limited dilution method. shRNA constructs used for mouse Sdcl knock down are as follows:

pMLP-Sdcl-shl:

5’ -CGGGGATGACTCTGACAACTTA-3’ (SEQ ID NO: 7),

5’ -TAGTGAAGCCACAGATGTA-3’ (SEQ ID NO: 8), and

5’ -TAAGTTGTCAGAGTCATCCCCA-3’ (SEQ ID NO: 9),

pMLP-Sdcl-sh2:

5’ - ACAGGCAGCTGTC AC ATCTCAA-3’ (SEQ ID NO: 10),

5’ -TAGTGAAGCCACAGATGTA-3’ (SEQ ID NO: 11), and 5’ -TTGAGATGTGACAGCTGCCTGG-3’ (SEQ ID NO: 12), and

pMLP-Sdcl-sh3:

5’ -CCAAGACTTCACCTTTGAAACA-3’ (SEQ ID NO: 13),

5’ -TAGTGAAGCCACAGATGTA-3’ (SEQ ID NO: 14), and

5’ -TGTTTC AAAGGTG AAGTCTTGT-3’ (SEQ ID NO: 15).

shRNA sequences used for mouse TLR4 knock down are as follows:

5’ -CACTTAGACCTC AGCTTCA ATGGTGCC AT-3’ (SEQ ID NO: 16) and

5’ -TGCCTTCACTACAGAGACTTTATTCCTGG-3’ (SEQ ID NO: 17).

Western blotting. Cells were lysed in RIPA lysis buffer (Millipore) and protein concentration was measured by BCA protein assay kit (Thermo Fisher Scientific). Samples were separated by SDS-PAGE and electroblotted to polyvinylidene fluoride (PVDF) membranes (GE Healthcare). In the case of SDC1 detection, cationic nylon membrane (Immobilon Ny; Millipore) was used. Signals were visualized with SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer's protocol. Chemiluminescence was quantitated using a Bio-Rad ChemiDoc™ MP imaging system.

Quantitative RT-PCR. Total RNA was extracted by TRIzol® (Life Technologies) and reverse transcribed into cDNA by using Superscript III reverse transcriptase (Life Technologies) according to the manufacturer's protocol. Analysis of mRNA levels was performed on a 7900 Fast Real-Time PCR System (Life technologies) with SYBR Green-based real-time PCR. The primer sequences used for real-time PCR are as follows:

(sense) 5’ -CTCAGAGCCTTTTGGACAGG-3’ (SEQ ID NO: 18) and

(antisense) 5’TACAGCATGAAAGCCACCAG-3’ (SEQ ID NO: 19) for mouse Sdcl

(sense) 5 '-TGTGTACACGGAGAAACATTCAG-3 ' (SEQ ID NO: 20) and

(antisense) 5 '-GCAAAGAGAAAGCCGATCAC-3 ' (SEQ ID NO: 21) for mouse Sdc2

(sense) 5 AACTGAGGTCTTGGCAGCTC-3’ (SEQ ID NO: 22) and

(antisense) 5’TACACCAGCAGCAGGATCAG-3’ (SEQ ID NO: 23) for mouse Sdc4

(sense) 5’ -CCAATTTTTCAGAACTTCAGTGG-3’ (SEQ ID NO: 24) and

(antisense) 5’ - AGAGGTGGTGTAAGCC ATGC 3’ (SEQ ID NO: 25) for mouse Tlr4

(sense) 5’ -GCTGATGCTGTCAAGCTGAG-3’ (SEQ ID NO: 26) and

(antisense) 5’ -GAGCCTCCTGTTTTGTCTCG-3’ (SEQ ID NO: 27) for mouse Caspase-ll

(sense) 5- CCTCTGTGAGGTGCTGAAAC-3’ (SEQ ID NO: 28) and

(antisense) 5’ -TCAGGCTTTTCTTCCTGGAG-3’ (SEQ ID NO: 29) for mouse Nlrp3\

(sense) 5’ -ACAAGACCCACGTGGAGAAG-3’ (SEQ ID NO: 30) and

(antisense) 5’ - AGCCC ATTGTGCTGTCTCTC-3’ (SEQ ID NO: 31) for human Caspase-4 (sense) 5’ TGGGCTGTTTAAAGTCCAGAAG-3’ (SEQ ID NO: 32) and

(antisense) 5’ -TTTGTTTTGCTTGGGTTTCC3’ (SEQ ID NO: 33) lor mouse Aim2

(sense) 5’ -ACATGGGCTTACAGGAGCTG-3’ (SEQ ID NO: 34) and

(antisense) 5’ - ACTCTGAGCAGGGACACTGG-3’ (SEQ ID NO: 35) lor mouse Aw;

(sense) 5’ -TGTCTGGTGCTTGACTCTGG-3’ (SEQ ID NO: 36) and

(antisense) 5’ -CTGGGTTTCACTCAGCACAG-3’ (SEQ ID NO: 37) for mouse Gsdmd

(sense) 5’ -GCTGTGACCCTCTCTGTGAAG-3’ (SEQ ID NO: 38) and

(antisense) 5’ -TTTCAGGTGGATCCATTTCC-3’ (SEQ ID NO: 39) for mouse 1118\

(sense) 5’ - AAAGCTCTCC ACCTC AATGG-3’ (SEQ ID NO: 40) and

(antisense) 5’- AGGCCACAGGTATTTTGTCG-3’ (SEQ ID NO: 41) for mouse Illb.

Co-immunoprecipitation assay. COS-l cells were transfected with 2.5 pg each of candidate gene cloned into pcDNA3.l/Myc-His vector, the human SCGB3A2 (NM_054023) open reading frame cloned into pcDNA3.l with a C-terminal FLAG tag, or a control plasmid by using X-tremeGene™ HP DNA transfection reagent (Roche Applied Science). Both cells and media were harvested 48 hours after transfection. The culture media containing cells were centrifuged at 500 g for 10 minutes at 4°C and the supernatant was collected (media supernatant). Cells were lysed in 400 pL CHAPS IP buffer-l (1% CHAPS, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, protease inhibitor complete-mini 1 tablet/lO ml) and sonicated two times for 5 seconds each on ice. The cell lysates were centrifuged at 15,000 g for 10 min at 4°C and the supernatant was collected (cell lysate supernatant). The media supernatant and cell lysate supernatant were combined, which were pre cleared with Protein G- Agarose (Santa Cruz Biotechnology) at 4°C for 3 hours, followed by incubation with FLAG-tagged gel (20 pL; #3326, MBL) at 4°C overnight. The gel- immunocomplexes were washed twice with CHAPS IP buffer-2 (0.1% CHAPS, 500 mM NaCl, 50 mM Tris-HCl, pH 7.4) for 20 min each and then washed twice with CHAPS IP buffer-3 (0.1% CHAPS, 50 mM Tris-HCl, pH 7.4) for 20 min each.

Immunoprecipitated samples were separated by SDS-PAGE and electroblotted to PVDF membranes. Blocking was carried out with 5% skim milk in TBST (Tris-buffered saline; Tris-HCl, pH 7.4 + 0.1% Tween 20) and the membrane was subsequently incubated with anti-Myc mouse monoclonal antibody (1:1000, 9B11, Cell signaling) at 4°C overnight followed by incubation with sheep anti-mouse IgG HRP-linked F(ab')₂ fragment (1:2000; NA9310, GE Healthcare). Signals were detected as described for western blotting.

Streptavidin pull down assay. LPS-Biotin (1 mg/rnl) and immobilized Streptavidin agarose gel were incubated for 30 min at 4 C. and after biotin blocking, 1.25 mg/ml recombinant human SCGB3A2 was added as a pray protein and incubated for 1 hr at 4°C. Ten % of flow through was used as an input. After washing several times, the gel was boiled for 5 min with SDS sample buffer and the supernatant was used for western blotting.

Imidazole-zinc staining. Imidazole-zinc staining was carried out as previously reported (Rodriguez and Hardy. Anal Biochem 485, 72-80, 2015). Briefly, LPS dissolved and/or SCGB3A2 diluted in water were loaded onto 0.8% agarose gel in full in a well to make sure the content reaching to gel surface and run at 50V in TAE buffer (Tris-acetate-EDTA buffer; 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA, pH 8.0) until dye reached to the gel bottom. The gel was washed with ddfhO and immersed in 0.2 M imidazole for 20 min with gentle agitation. After discarding solution and washing with ddH20, the gel was placed in the dark and incubated with 0.3 N zinc sulfate solution for several minutes. Then the gel was rinsed with ddH20 to stop staining and an image was taken with ChemiDoc™ imaging system (Bio-Rad). For double staining experiments, the gel was stained with 0.25% Coomassie Brilliant Blue solution after the gel image of Imidazole-zinc staining was scanned.

FACS analysis. For LPS and cell binding assay, cells were washed with PBS and incubated with Alexa 488 or 594-conjugated LPS from E. coli 055:B4 (L-23351 or L-22353, 1 pg) (Thermo Fisher Scientific) with or without SCGB3A2 (1 pg/ml) at 4°C for 30 min. After washing with PBS, the cells were analyzed in a FACS Canto II (Becton Dickinson). For the SCGB3A2 and LLC cell binding assay, LLC cells were incubated with recombinant mouse or human SCGB3A2, washed with PBS, incubated with anti-SCGB3A2 antibody for 30 min followed by PE-rabbit IgG secondary antibody for 30 min. For SDC1 expression analysis, LLC cells were harvested in PBS and stained with PE-rat anti-mouse SDC1 (clone 281.2, BD Pharmingen) for 30 min at 4°C. For Annexin V/PI analysis, Dead Cell Apoptosis Kit with Annexin V FITC and PI, for flow cytometry (V13242, Thermo Fisher Scientific) was used. Cells were harvested using a scraper and washed with cold PBS and stained with Annexin V- Alexa 488 and PI in lx Annexin binding buffer for 15 minutes. As a compensation control, FITC-stained only or PI stained only cells were prepared by inducing cell death by incubation in 70% EtOH for 10 minutes. All experiments were carried out in the NCI Flow Cytometry Core Facility.

Immunofluorescence analysis. All adherent cells were seeded on glass coverslips (Nunc™ Lab-Tek™ Chambered Coverglass (15583PK, Nunc). Floating cells in culture were subjected to cytospin (Shandon) before further processing. After fixation with 10% buffered formalin for 10 minutes at room temperature (RT), cells were permeabilized with 100% MeOH at - 20°C for 10 minutes. Blocking was done with 1% BSA in PBS for 1 hour and cells were stained with primary antibodies for 1 hour at RT. After wash with PBS, cells were stained with secondary antibodies (1:200, Alexa flour, Molecular Probe) for 45 min at RT. Cells were analyzed under confocal microscope (Zeiss 510/710) according to the NCI confocal microscope facility manual or Keyence microscope BZ-X700. For human cell lines, adherent cells staining was same with above procedures. For floating cells, supernatants were immobilized on glass slides (Shandon™ Double Cytoslides™, Thermo Fisher Scientific) using cytospin centrifugation and stayed at RT overnight. After fix with 10% formalin, performed same immunofluorescence protocol described above.

LLC cells mouse metastasis model. LLC cells (2xl0⁵ cells) were intravenously administered to C57BL/6N mice (Charles River, Frederick, MD), followed by daily intravenous administration of recombinant mouse or human SCGB3A2 (0.25 mg/kg/day) for 7 days starting at day 0 (30 min after LLC cells injection), 7, or 14 or during the entire experimental period of 20 days, or PBS injection for 20 days as control. Mice were killed on day 21 and the numbers of lung metastasized tumors evaluated. Some lungs were subjected to histological analysis. All animal studies were carried out after approval by the National Cancer Institute Animal Care and Use Committee.

Histological analysis. Lung samples were fixed in 10% buffered formalin under 20-cm FLO pressure, embedded in paraffin, sectioned at 4 pm by microtome and performed with

Hematoxylin and Eosin staining (H&E).

Lung carcinogenesis study. Ccsp-Cre;LSL-Kras^G12D conditional mutant mice on the 129SvJ-C57BL/6 mixed background (Jackson et al, Genes Dev 15:3243-3248, 2001; Moghaddam et al, Am J Respir Cell Mol Biol 40:443-453, 2009) express the oncogenic Kras^Gl2D gene in lung- specific fashion. Scgb3a2(fl/fl ) mice, previously described (Kido et al. , Mediators Injlamrn 2014: 1- 10, 2014), were backcrossed to C57BL/6N mice three times. Ccsp-Cre;LSL- Kras^G12D and Scgb3a2(fl/fl) mice were crossed to produce Ccsp-Cre;LSL- Kras^G12D;Scgb3a2(jl/jl) (tentatively named Kras^G12D;Scgb3a2(fl/jl )) and littermate Ccsp-Cre;LSL- Kras^G12D ;Sc b3(jl/+ ) (tentatively named Kras^GI2D ;Scgb3a2(fl/ +)) mice, and male mice were used in the study. Mice were maintained under standard specific-pathogen-free conditions.

HaloTag imaging. To construct a HaloTag-mouse SCGB3A2 (mSCGB3A2-HT) expression vector, pFC14A HaloTag® CMV Flexi® Vector (Promega) was fused to C-terminal of mouse SCGB3A2 cDNA. Primers for the SCGB3A2 HaloTag plasmid were designed using the Flexi® Vector Primer Design Tool web site. A HaloTag® Coding Region Control Expression Vector (Control-HT) was designed according to the manufacture’s instruction. mSCGB3A2-HT or Control-HT was transfected to HEK293 cells using X-tremeGENE™ HP DNA Transfection Reagent and after 48 or 72 hours, supernatant was collected and concentrated with Amicon Ultra (Millipore) and stored at -80°C until use. The transfection efficiency was confirmed with microscopy using TMR Direct HaloTag ligand. For uptake of HT-mSCGB3A2 into LLC cells, after addition of HT-mSCGB3A2, cells were stained with HaloTag®TMR ligand for short incubation time or HaloTag® TMRDirect™ ligand overnight. After two washes with PBS, the cells were visualized under a microscope.

DLS. Dynamic light scattering analysis (DLS) was performed using DynaPro Nanostar (Wyatt). The radius of LPS-SCGB3A2 complex was determined after samples were centrifuged and dissolved in 50 pL of 0.22 pm filtered sterile PBS. The evaluation of data was performed by Dynamics V7 software.

Limulus Amebocyte Lysate (LAL endotoxin) assay. LPS quantification in each

SCGB3A2 recombinant protein was performed using the ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (L00350, GenScript).

LDH assay. RAW264.7 cells grown in 96 flat bottom well plates supplemented with OPTI-MEM (31985062, Thermo Fisher Scientific) were incubated with or without SCGB3A2 and/or LPS (011LB4) in the media for 16 hours. Lysed cell supernatants were evaluated for the presence of cytoplasmic enzyme lactate dehydrogenase (LDH) using the Pierce™ LDH

Cytotoxicity Assay Kit (Thermo Fisher Scientific). Cytotoxicity was calculated according to the kit instructions; as a percentage of (experimental LDH - spontaneous LDH)/(maximum LDH release - spontaneous LDH).

TUNEL assay. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) analysis was performed using DeadEnd™ Fluorometric TUNEL System (G3250, Promega) according to the manufacturer’s instructions. Total tumor areas and TUNEL positive areas were measured using imageJ software, and a percentage of TUNEL positive areas per total tumor areas was calculated.

SCGB3A2 modeling. A SCGB3A2 dimer model was build starting from a consensus secondary structure prediction obtained using several procedures including I-TASSER

(zhanglab.ccmb.med.umich.edu/ITASSER/); LOMETS

(zhanglab.ccmb.med.umich.edu/LOMETS/); RaptorX (raptorx.uchicago.edu); Swissmodel (swissmodel.expasy.org); Phyre2 (www.sbg.bio.ic.ac.uk/phyre2 ); BHAGEERATH-H

(scfbioiitd.res.in/bhageerath/bhageerath_h-jsp) and Quark

(zhanglab.ccmb.med.umich.edu/QUARK ). The above-mentioned procedures were used as available in their respective web- site implementations as of March 2017. The methods explored span the spectrum of structure prediction techniques including threading, library based methods, etc. None of the methods explored produced a compact structure. The helical motifs were properly identified by all models. The consensus helical regions as described in FIG. 11D were manually aligned against the uteroglobin structure (PDB ID: 1UTG) identified as the closest homolog of SCGB3A2 for which an experimental structure is currently available. The missing sections connecting the helical motifs were modeled as loops to the sole purpose of connecting the helices in an initial workable model. The model was then refined using Feedback Restrain Molecular Dynamics (FRMD). FRMD is based on a self-consistent procedure to bias molecular dynamics trajectories towards a refined conformation using experimental information from multiple sources including X-ray diffraction or NMR data when available (Cachau et al, Cell Mol Biol (Noisy-le- grand) 49, 973-983, 2003; Cachau et al, International J High Performance Computing Applications 8, 24-34, 1994; Cachau et al, Protein Eng 7, 831-839, 1994). The procedure is conceptually similar to a reversed molecular replacement protocol when using X-ray data, with the additional advantage that only those regions of the molecule in agreement with the crystallographic data are affected by the crystallographic constrain, as weighted by the FRMD protocol thus preserving the structural homology when available (Cachau et al , International J High Performance Computing Applications 8, 24-34, 1994). FRMD was implemented in QMRx (Fadel et al Acta Crystallogr D Biol Crystallogr 71, 1455-1470, 2015) using Xplor-NIH (Schwieters et al, J Magn Reson 160, 65- 73, 2003) to compute the crystallographic restrains and GROMACS 5.1.4 (Abraham et al,

SoftwareX 1-2, 19-25, 2015) to drive the molecular dynamics (MD) calculations using the Amber ff99sbildn force field for all MD calculations. All calculations were performed using a time step of 2 fs. All bonds were constrained for all MD calculations. The leapfrog algorithm was used for integration using a velocity rescaling thermostat (Noose-Hover) with a 0.1 ps coupling constant. Electrostatic forces were computed using a distance criteria, and a cutoff of 10 A was used for van der Waals interactions. No periodic boundary conditions were used aside from the periodicity resulting from the X-ray constrains. The system was freely equilibrated at T = 300 K for 5 ns without constrains, the purpose of this short run was to relax the initial model without losing the original shape of the model. The model was then fully relaxed using FRMD with X-ray restrains as described in (Cachau et al, International J High Performance Computing Applications 8, 24-34, 1994) and Fcalc values computed for PDB ID: 1UTG in-lieu of experimental values not deposited for this entry in the Protein Data Bank, and limited to a 6 A resolution cutoff. The nature of the FRMD procedure restricts the value of energy based monitors. The convergence of the model was monitored using a crystallographic R factor and RMSD (root mean square deviation) against the reference structure for homologous residues (see FIG. 11D). The trajectory converges to the structure shown in FIG. 11 after 350 ns with an R value of 9.3 (6 A) and RMSD 3.2 A. The MD trajectory was continued for another 350 ns without noticeable changes in the structure. The dimer structure was used to explore possible tetrameric arrangements by rolling a dimer against another using GROMACS and the AMBER force field to probe the interaction. A favorable arrangement was detected as described in FIG. 11N. The number and placement of Cys in 1UTG and SCGB3A2 are different. Thus, SCGB3A2 was modeled replacing Cys 48 by Ala to avoid the possible bias that could have resulted from imposing a disulfide bond during the MD calculation. Ala 48 was then replaced back to Cys in the final dimer model where the two Cys S atoms appear at less than 2.5A from each other suggesting a proper placement of the Cys 48 in the dimer. FRMD can be used to estimate the data lost during the modeling procedure by reversing the refinement procedure i.e. 1UTG was modeled from the final model of SCGB3A2 using an identical protocol as previously used to model SCGB3A2 from 1UTG. The structure of 1UTG thus modeled agrees with the experimental one with an RMSD 3.5A (backbone atoms).

Statistical analysis. Statistical analysis was carried out using GraphPad Prism v7. Data are shown as means ± SD. Levels of significance for comparison between samples were determined by student’s t-test or one-way ANOVA. For the lung carcinogenesis study, the Kaplan-Meier method was used to estimate survival rates of mice and the log-rank (Mantel-Cox) test for comparing survival differences between groups. P values of <0.05 were considered statistically significant.

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described embodiments. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

It is claimed:

1. A method of treating or preventing cancer in a subject, comprising:

administering to a subject with cancer or at risk of cancer a therapeutically effective amount of lipopolysaccharide (LPS) and secretoglobin 3A2 (SCGB3A2) protein to inhibit the cancer in the subject; or

administering to a subject with cancer or at risk of cancer a therapeutically effective amount of a secretoglobin 3A2 (SCGB3A2) protein without LPS to inhibit the cancer in the subject, wherein the cancer is not lung cancer.

2. The method of claim 1, wherein the cancer is a syndecan-l (SDC1) positive cancer.

3. The method of claim 1 or claim 2, wherein the cancer expresses a non-canonical inflammasome.

4. The method of any one of claims 1-3, wherein cells of the cancer express CASP4 and/or CASP5.

5. The method of any one of claims 1-4, wherein the cancer is an epithelial cell cancer.

6. The method of any one of claims 1-5, wherein the cancer is a colon cancer.

7. The method of any one of claims 1-6, comprising administrating the therapeutically effective amount of LPS and SCGB3A2 protein to inhibit the cancer in the subject, wherein the cancer is a lung cancer.

8. The method of claim 7, wherein the lung cancer is a small-cell lung carcinoma (SCLC) or a non- small-cell lung carcinoma (NSCLC)/

9. The method of claim 8, wherein the NSCLC is adenocarcinoma, squamous cell carcinoma, large cell carcinoma or bronchioalveolar carcinoma.

10. The method of any one of claims 1-9, wherein treating the cancer in the subject reduces tumor metastasis.

11. The method of claim 10, wherein reducing metastasis of the cancer reduces tumor metastasis to the lung of the subject.

12. The method of any one of claims 1-11, wherein treating the cancer reduces growth of the cancer.

13. The method of any one of claims 1-12, wherein treating the cancer reduces proliferation of the cancer.

14. The method of any one of claims 1-13, wherein treating the cancer reduces tumor burden in the subject.

15. The method of any one of claims 1-14, wherein the subject is a human.

16. The method of any one of claims 1-15, wherein the SCGB3A2 protein is human SCGB3A2 protein.

17. The method of any one of claims 1-16, wherein the SCGB3A2 protein comprises or consists of an amino acid sequence at least 90% identical to the amino acid sequence set forth as SEQ ID NO: 1 and binds to LPS and SDC1.

18. The method of claim 17, wherein the SCGB3A2 protein comprises or consists of the amino acid sequence set forth as SEQ ID NO: 1.

19. The method of any one of claims 1-18, wherein the LPS and SCGB3A2 protein are administered intravenously, intra-tumorally, locally to a tumor, orally, by injection into the intestine, or by inhalation.

20. The method of any one of claims 1-19, wherein the therapeutically effective amount the SCGB3A2 protein and the LPS comprise:

from about 0.005 mg SCGB3A2/kg bodyweight to about 500 mg SCGB3A2/kg bodyweight; and

from about 0.05 EU/kg body weight to about 5 EU/kg body weight.

21. The method of any one of claims 1-20, further comprising selecting the subject with the cancer or at risk of the cancer prior to administration of the therapeutically effective amount of the LPS and the SCGB3A3 protein, or the SCGB3A3 protein, wherein the cancer is not lung cancer.

22. The method of any one of claims 1-21, further comprising administering to the subject a therapeutically effective amount of an additional anti-tumor agent.

23. Use of SCGB3A2 and LPS to inhibit growth and/or proliferation of a tumor in a subject.

24. A pharmaceutical composition formulated for administration to a subject, comprising LPS, human SCGB3A2 protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1, and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24, wherein the human SCGB3A2 protein comprises or consists of the amino acid sequence set forth as SEQ ID NO: 1.

26. The composition of claim 24 or claim 25, comprising:

from about 1 pg/ml to about 1 mg/ml SCGB3A2 protein; and

from more than 0.01 EU/ml to about 5 EU/ml LPS.

27. The composition of any one of claims 24-26, formulated for oral administration and delayed release in the intestine.

28. The composition of any one of claims 24-26, formulated for administration by inhalation.