Fig 1: PTHrP differentially regulates p27 through LIFR in breast cancer cells. (A) Immunofluorescence staining and quantification of LIFR in primary tumors from mice inoculated with MSCV, FLSEC, DNLS, or DNLS + CTERM cells. All panels = 40X and scale bars = 50 μm. (B) Western blot analysis of p27, pERK, ERK, p-p38, p38 and tubulin (loading control) protein levels in MSCV, FLSEC, DNLS, or DNLS + CTERM cells treated with vehicle (DMSO) or LIFR inhibitor (EC359, 50nM or 100nM) for 24 h. Densitometry for western blot analysis of (C) p27, (D) pERK/ERK and (E) p-p38/p38 described in (B). (A) **p < 0.01 vs. DNLS by unpaired t-test. (C) *p < 0.05 vs. DNLS by unpaired t-test or *p < 0.05 vs. MSCV by one-way ANOVA with multiple comparisons. (D & E) *p < 0.05 vs. MSCV by one-way ANOVA with multiple comparisons or *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle by two-way ANOVA. Graphs represent mean ± SEM
Fig 2: Validation of plasmids expressing specific PTHrP domains. (A) Pthlh overexpression construct design and validation in MCF7 cells by (B) western blot for the C-terminal HA-Tag and qPCR for the (C) mid-region, (D) nuclear localization sequence (NLS), and (E) C-terminal domain. MSCV = control, FLSEC = full-length secreted, DNLS = NLS deleted, DNLS + CTERM = NLS and C-terminal domain deleted. Predicted molecular weights: FLSEC PTHrP (-36-139aa) = 21.2kD, DNLS PTHrP (-36-67aa)…(95-139aa) = 18kD, DNLS + CTERM PTHrP (-36-67aa) = 12.8kD. GAPDH = loading control. (F) Immunocytochemical staining for HA-Tag (green) and DAPI (blue). All panels = 100X and scale bars = 25 μm. (G) Secreted PTHrP (1-34aa) levels measured by ELISA from conditioned media of cells described in (A). (B-E & G) n = 3 independent biological replicates. Graphs represent mean ± SEM. (C) **p < 0.001 vs. MSCV or *p < 0.05 vs. FLSEC by one-way ANOVA with multiple comparisons. (D) **p < 0.001 vs. FLSEC by one-way ANOVA with multiple comparisons. (E) **p < 0.001 vs. DNLS by one-way ANOVA with multiple comparisons
Fig 3: Model of PTHrP domain-specific actions in breast cancer progression and bone colonization. In the primary breast site (top left panel, left of arrows), PTHrP lacking the NLS and C-terminal domain decreases tumor cell proliferation through p27 induction driven by the tumor suppressor leukemia inhibitory factor receptor (LIFR). PTHrP lacking the NLS and C-terminal domain also preferentially induces p38 phosphorylation and signaling to inhibit cell cycling downstream of LIFR activation. In the breast, truncated PTHrP lacking the NLS alone (top left panel, right of arrows) downregulates LIFR expression (denoted by transparent coloring) and prevents induction of p27 expression and activation of p38 signaling (denoted by dashed arrows, dotted outlines and transparent coloring) to drive cell proliferation and tumor growth. In bone disseminated tumor cells (bottom panel), LIFR expression is downregulated and the induction of p27 by PTHrP lacking the NLS and C-terminal domain persists, but is not sufficient to repress metastatic outgrowth (denoted by dashed inhibitor line), in contrast to the primary tumor. In the bone, tumor cells expressing PTHrP peptides lacking the NLS or NLS and C-terminal domain readily proliferate into metastatic tumors. Image created with Biorender.com
Fig 4: Truncated. PTHrP induces CDKN1B in the bone metastatic site, but enhances osteolysis and tumor burden. (A) qPCR analysis for CDKN1B (p27) normalized to ACTB as a marker of total tumor burden in the bone marrow of mice inoculated with MSCV, FLSEC, DNLS, or DNLS + CTERM cells via intracardiac injection. n = 8–10 mice/group. (B-D) Total osteolytic lesion area and lesion number (per mouse) based on radiographic analyses for mice described in A. White arrows indicate osteolytic lesions. (E) Flow cytometric quantitation of percent CD298 + tumor cells in the bone marrow of mice described in A. (F) qPCR analysis for RANKL/OPG (Tnfsf11 / Tnfrsf11b) in whole homogenized femurs from mice described in (A). n = 8–10 mice/group. *p < 0.05, **p < 0.01, or ****p < 0.0001 vs. MSCV by one-way with multiple comparisons. Graphs represent mean ± SEM
Fig 5: Deletion of the PTHrP NLS alters breast cancer cell proliferation and primary tumor growth. (A) Time to tumor palpation, (B) tumor volume over time by digital caliper measurement and (C) final tumor weight in mice inoculated with MSCV, FLSEC, DNLS, or DNLS + CTERM cells into the mammary fat pad. n = 7–10 mice/group. (D) Ki67 staining and quantification from tumors in (A-C). (E) Quantification of mitoses (# mitotic figures/total cells in 40X field) by DAPI staining from tumors in (A-C). (F) Cleaved PARP staining and quantification from tumors in (A-C). All panels = 40X and scale bar = 50 μm. (A) *p < 0.05 vs. MSCV by one-way ANOVA with multiple comparisons or #p < 0.05 DNLS vs. DNLS + CTERM by unpaired t-test. (B) ****p < 0.0001 vs. MSCV by one-way ANOVA with multiple comparisons or **p < 0.01 vs. DNLS by unpaired t-test. (C) **p < 0.01 vs. MSCV by one-way ANOVA with multiple comparisons or ***p < 0.001 vs. DNLS by unpaired t-test. (D) **p < 0.01 vs. MSCV by one-way ANOVA with multiple comparisons. (E) *p < 0.05 vs. MSCV by one-way ANOVA with multiple comparisons or *p < 0.05 vs. DNLS by unpaired t-test. Graphs represent mean ± SEM
Supplier Page from Creative Diagnostics for PTH-RP ( 1-34 ) ( human, rat, mouse ) ELISA Kit