Tumor-derived osteopontin isoforms cooperate with TRP53 and CCL2 to promote lung metastasis

Abstract

The lungs are ubiquitous receptacles of metastases originating from various bodily tumors. Although osteopontin (SPP1) has been associated with tumor dissemination, the role of its isoforms in lung-directed metastasis is incompletely understood. We employed syngeneic mouse models of spontaneous and induced lung-targeted metastasis in C57BL/6 mice competent and deficient in both Spp1 alleles. Tumor-derived osteopontin expression was modulated using either stable anti-Spp1 RNA interference, or forced overexpression of intracellular and secreted Spp1 isoforms. Identified osteopontin's downstream partners were validated using lung adenocarcinoma cells conditionally lacking the Trp53 gene and Ccr2-deficient mice. We determined that host-derived osteopontin was dispensable for pulmonary colonization by different tumor types. Oppositely, tumor-originated intracellular osteopontin promoted tumor cell survival by preventing tumor-related protein 53-mediated apoptosis, while the secretory osteopontin functioned in a paracrine mode to accelerate lung metastasis by enhancing tumor-derived C-C-motif chemokine ligand 2 signaling to cognate host receptors. As new ways to target osteopontin signaling are becoming available, the cytokine may constitute an important therapeutic target against pulmonary involvement by cancers of other organs.

Keywords: C–C-motif chemokine ligand 2; inflammation and cancer; secreted phosphoprotein 1; spontaneous lung metastasis; tumor-related protein 53.

Figure 1.

Characterization of osteopontin transcript protein products and impact of host-expressed SPP1 on lung metastasis. (A) Immunoblots probed with anti-SPP1 and anti-GFP antibodies of whole cell protein extracts of HEK293T cells (293T) transfected with no vector or with bicistronic vectors encoding Spp1is2 or Spp1is4 in-frame with GFP or GFP alone. Note the correspondence of the Spp1is2 transcript to the iSPP1.GFP fusion protein that runs at ∼68 KDa and of the Spp1is4 transcript to the sSPP1.GFP fusion protein that runs at ∼50 KDa, the GFP protein at ∼27 KDa, as well as the ∼40 KDa band appearing only after Spp1is4 transfection, which likely represents a proteolytic fragment of osteopontin. (B) Representative images of immunostaining of the main cell types and anatomic compartments of the naïve C57BL/6 mouse lung (n = 5) for endogenous SPP1 (brown), counterstained with hematoxylin (blue). (C, D) Primary tumor growth rate and number and total volume of lung metastases of Spp1-competent (Spp1^+/+) and Spp1-deficient (Spp1^−/−) mice after (C) s.c. injection (n = 5–7/group) or (D) i.v injection (n = 6–15/group) of B16F10, LLC, or MC38 cells. Note that only mice that received s.c LLC cells developed spontaneous lung metastases. Data are expressed as mean ± SD ns and *p > 0.05 and p < 0.05, respectively, for comparison between Spp1^+/+ and Spp1^−/− mice by two-way ANOVA with Bonferroni post-tests (dot plots) or unpaired Student's t-test (bar graphs).

Figure 2.

SPP1 isoform expression by C57BL/6 tumor cell lines and their role in cancer cell survival. (A–D) Parental B16F10, LLC, and MC38 cells, B16F10 cells stably expressing Spp1is2 and Spp1is4 constructs (p), and LLC and MC38 cells stably expressing anti-Spp1 (shSpp1) or random control (shC) shRNA were assessed for Spp1 mRNA by RT-PCR of total cellular RNA (A),SPP1 protein by immunoblotting of whole cells extracts (B), ELISA of cellular supernatants (C) and SPP1 cellular immunostaining (D). Inlays: isotype controls. Note amplicons of Spp1is2 and Spp1is4 at 933 and 885 bp, respectively, and the corresponding electrophoretic bands of iSPP1 and sSPP1 at 41 and 23 KDa (A and B). Note also that sSPP1 displays an apparent molecular mass that is smaller than anticipated by Spp1is4 sequence due to post-translational modification (cleavage) (B). Finally, note that only sSPP1 was detected in media conditioned for 24 h by 10⁵ live cells (n = 5/group) (C). (E–G) Parental and SPP1-modulated tumor cells described above were assessed for cellular proliferation by MTT reduction (E) and for apoptosis by flow cytometric determination of annexin V and 7AAD staining (F) and caspase 3 and 8 immunoreactivity by immunocytochemistry (G). Data are expressed as mean ± SD of one representative of three experiments performed both in the presence or absence of 10% FBS. ns, **, and ***p > 0.05, p < 0.01, and p < 0.001, respectively, for the indicated comparisons by two-way (dot plots) or one-way (bar graphs) ANOVA with Bonferroni post-tests.

Figure 3.

A murine lung adenocarcinoma cell line conditional for Trp53 alleles reveals that Trp53 is required for the pro-survival effects of intracellular SPP1. (A) Strategy for derivation of C57BL/6 Urethane-induced Lung Adenocarcinoma cells (CULA) from mice carrying loxP sites on either side of both Trp53 alleles (Trp53^f/f). Mice (n = 5) were initiated on 10 weekly intraperitoneal urethane injections (1 gr/kg) 6 weeks after birth and were sacrificed 10 months after the first injection. Lungs were harvested under sterile conditions, tumors (n = 10) were enucleated, minced, and cultured separately. One clone was established after 4 weeks and was passaged more than 100 times over 2 y. (B) Representative stereoscopic image of lungs from Trp53^f/f C57BL/6 mouse treated as described under (A) featuring lung tumors (arrows). (C, D) Representative images of immunostaining of lung tissue from Trp53^f/f C57BL/6 mouse treated as described under (A) for PCNA, shows minimal nuclear immunoreactivity in airway epithelium (arrows in C) in contrast to the significant proportion of immunoreactive cells (arrows) in urethane-induced tumors (dashed outline in D). (E) Phase contrast image of Trp53^f/f CULA cells in culture at passage 40. Note the spindle-shaped tumor cells. (F–I) C57BL/6 mice (n = 9) received 0.5×10⁶ Trp53^f/f CULA cells s.c. and were sacrificed after 8 weeks. Representative images of flank tumor [dashed outline; (F)], spontaneous lung metastases [arrows; (G)], lung section with lung metastases [(H); arrows], and of lung section with lung metastasis showing several mitoses per high-power field [arrows; (I)]. (J) RT-PCR of Trp53^f/f CULA cells stably expressing vectors encoding a random sequence (pC) or CRE recombinase (pCre). Successful deletion of floxed Trp53 alleles is confirmed by the 270 bp product. (K) Immunoblots of whole cell protein extracts of Trp53^f/f CULA cells stably expressing vectors encoding a random sequence (pC) or CRE recombinase (pCre) and random control (shC) or Spp1-specific shRNA (shSpp1) showing efficient combined modulation of both TRP53 and SPP1 protein expression in this system. (L, M) CULA cells featuring conditional Trp53 alleles (Trp53f/f) were stably transfected as described in (K) were assessed for cellular proliferation by MTT reduction (L) and for apoptosis by flow cytometric determination of annexin V and 7AAD staining (M). Data are expressed as mean ± SD (n = 5/data-point) of one representative of three experiments performed both in the presence or absence of 10% FBS. ns, *, **, and ***p > 0.05, p < 0.05, p < 0.01, and p < 0.001, respectively, for the indicated comparisons by two-way (dot plots) or one-way (bar graphs) ANOVA with Bonferroni post-tests.

Figure 4.

Tumor-derived SPP1 in spontaneous lung metastasis. Primary tumor growth rate (A–C), survival (D), and number (E), total volume (F), and representative images (G) of lung metastases of C57BL/6 mice (n = 6–16/group) after s.c. injection of parental and SPP1-modulated tumor cells described in Fig. 2. Note that only mice that received LLC cells developed spontaneous lung metastases. Shown are mean ± SD (all graphs except D) or Kaplan–Meier survival estimates (D). ns and ***p > 0.05 and p < 0.001, respectively, for the indicated comparisons by two-way ANOVA with Bonferroni post-tests (dot plots), unpaired Student's t-test (bar graphs), or log-rank test (D).

Figure 5.

Tumor-derived SPP1 in induced lung metastasis. (A, B) Number and total volume of lung metastases (A) and survival (B) of C57BL/6 mice (n = 5–24/group) after i.v. injection of parental and SPP1-modulated tumor cells described in Fig. 2. (C) Representative microscopic images of lung sections stained with hematoxylin and eosin and stereoscopic images of lungs. Shown are mean ± SD (A) or Kaplan–Meier survival estimates (B). ns, *, **, and ***p > 0.05, p < 0.05, p < 0.01, and p < 0.001, respectively, for the indicated comparisons by one-way ANOVA with Bonferroni post-tests [(A), B16F10 cells], unpaired Student's t-test [(A), LLC and MC38 cells], or log-rank test (B).

Figure 6.

A requirement for CCL2 in the pro-metastatic effects of sSPP1. (A) Schematic of differential global gene expression by microarray of LLC and MC38 cells stably expressing anti-Spp1-specific (shSpp1) or random control (shC) shRNA. Listed are the top-10 common SPP1-dependent transcripts by order of magnitude. (B) qPCR of total cellular RNA of parental and SPP1-modulated tumor cells described in Fig. 2 for Ccl2 and Ccl7 relative to Gusb mRNA (n = 3/data-point). Shown is one representative of three experiments. (C) qPCR of total cellular and tissue RNA of parental tumor cells used in this study and of common target organs of metastasis for Ccl2 and Ccr2 relative to Gusb mRNA (n = 5/group). (D, E) Number of lung metastases of C57BL/6 mice competent (Ccr2^+/+) or deficient (Ccr2^−/−) in both Ccr2 alleles after i.v. injection of parental and SPP1-modulated MC38 (D) and B16F10 (E) cells described in Fig. 2 (n = 4–9/group). Data are expressed as mean ± SD ns, *, **, and ***p > 0.05, p < 0.05, p < 0.01, and p < 0.001, respectively, for the indicated comparisons (or comparison to brain in C) by one-way ANOVA with Bonferroni post-tests (B-D) or unpaired Student's t-test (E).