PTHrP drives breast tumor initiation, progression, and metastasis in mice and is a potential therapy target - PubMed (original) (raw)

. 2011 Dec;121(12):4655-69.

doi: 10.1172/JCI46134. Epub 2011 Nov 7.

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PTHrP drives breast tumor initiation, progression, and metastasis in mice and is a potential therapy target

Jiarong Li et al. J Clin Invest. 2011 Dec.

Abstract

Parathyroid hormone-related protein (PTHrP) is a secreted factor expressed in almost all normal fetal and adult tissues. It is involved in a wide range of developmental and physiological processes, including serum calcium regulation. PTHrP is also associated with the progression of skeletal metastases, and its dysregulated expression in advanced cancers causes malignancy-associated hypercalcemia. Although PTHrP is frequently expressed by breast tumors and other solid cancers, its effects on tumor progression are unclear. Here, we demonstrate in mice pleiotropic involvement of PTHrP in key steps of breast cancer - it influences the initiation and progression of primary tumors and metastases. Pthrp ablation in the mammary epithelium of the PyMT-MMTV breast cancer mouse model caused a delay in primary tumor initiation, inhibited tumor progression, and reduced metastasis to distal sites. Mechanistically, it reduced expression of molecular markers of cell proliferation (Ki67) and angiogenesis (factor VIII), antiapoptotic factor Bcl-2, cell-cycle progression regulator cyclin D1, and survival factor AKT1. PTHrP also influenced expression of the adhesion factor CXCR4, and coexpression of PTHrP and CXCR4 was crucial for metastatic spread. Importantly, PTHrP-specific neutralizing antibodies slowed the progression and metastasis of human breast cancer xenografts. Our data identify what we believe to be new functions for PTHrP in several key steps of breast cancer and suggest that PTHrP may constitute a novel target for therapeutic intervention.

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Figures

Figure 1

Figure 1. Cre-mediated Pthrp ablation in ME allows normal mammary development.

(A) Confocal images of IF staining with anti-PTHrP antibody in spontaneous breast tumors from standard PyMT mice and Western blot quantification showing increasing PTHrP expression in these tumors with respect to age. (B) Confocal images of IF staining for DAPI (blue) and PTHrP (green) in tumor tissues from control (Pthrpflox/flox;Cre– and PthrpWT;Cre+), heterozygous (Pthrpflox/+;Cre+), and homozygous (Pthrpflox/flox;Cre+) transgenic animals showing incremental decrease in PTHrP expression with allele ablation. Lower panel: Western blot quantification for PTHrP expression in tumors from the various genotypes. (C) Confocal images of IF staining with DAPI (blue), Cre recombinase (green), and PTHrP (red) showing colocalization of Cre and PTHrP. (D) Whole-mount staining analysis (Neutral Red) of mammary glands showing ductal outgrowth at 3, 5, and 7 weeks for control, heterozygous, and homozygous female virgin mice. (E) Western blot of PTHrP expression in mammary glands of virgin 7-week-old mice. Scale bars: 50 μm (A–C); 5 μm (D).

Figure 2

Figure 2. Pthrp ablation delays breast cancer initiation and progression.

(A) H&E staining of breast tissues from control (Pthrpflox/flox;Cre–) and homozygous (Pthrpflox/flox;Cre+) animals at 27, 35, and 45 days. Hyperplasias (arrowheads) are not detectable in homozygous animals before 45 days. Scale bars: 200 μm. (B) Tumor volume over time for control (Pthrpflox/flox;Cre– and PthrpWT;Cre+) and homozygous mice (Pthrpflox/flox;Cre+) showing that the delay in tumor growth is not related to the expression of the Cre gene. Inset: early time points. Values shown represent mean ± SD, n = 12 mice per group. ***P < 0.001. (C) Tumor weight per mouse at sacrifice (13 weeks) for control and homozygous animals. Values shown represent mean ± SD, n = 12 mice per group. ***P < 0.001. (D) Kaplan-Meier analysis of tumor onset for mice of all genotypes illustrating allelic effect for Pthrp ablation. (E) Kaplan-Meier analysis showing that control mice reach age requiring sacrifice much earlier than homozygous animals.

Figure 3

Figure 3. Pthrp ablation reduces spontaneous tumor load.

(A) Tumor load in whole animals (13 weeks). (B) Average weight of tumor load per mouse, average weight of primary tumors, and average number of tumors per mouse at sacrifice for the same genotypes. Values shown represent mean ± SD; n = 20 for Pthrpflox/flox;Cre–, n = 18 for Pthrpflox/+;Cre–, n = 22 for PthrpWT/WT;Cre+ (controls), n = 12 for Pthrpflox/+;Cre+ (heterozygous), and n = 30 for Pthrpflox/flox;Cre+ (homozygous), **P < 0.01.

Figure 4

Figure 4. A more complete ablation of Pthrp by Cre-carrying adenovirus further delays breast cancer initiation and progression.

(A) Adenovirus-transfected tumor cells selected by flow cytometry: left, cell fluorescence for GFP; right, confocal images of IF staining for DAPI (blue) and PTHrP (green) in mammary tumors derived from injected adenovirus-transfected tumor cells illustrating near-complete disappearance of PTHrP expression in homozygous tumors. Scale bars: 200 μm. (B) Western blot quantifying PTHrP expression in Pthrpflox/flox tumor cells transfected with adenoGFP. Lane 1, control, Pthrpflox/flox adenoGFP; lanes 2 and 3, hetero- and homozygous, Pthrpflox/+ or Pthrpflox/flox adenoCreGFP, respectively. (C) Tumor volume per animal for tumors derived from adenovirus-transfected tumor cells injected into the MFPs of syngeneic mice. Values represent mean ± SD, n = 12 mice for each group. ***P < 0.001. (D) Tumor load in whole animals 8 weeks after adenovirus-transfected cell injection. (E) Average weight of breast tumor load per mouse at sacrifice. Values represent mean ± SD, n = 12 mice per group. **P < 0.01.

Figure 5

Figure 5. Pthrp ablation modifies cell-cycle, apoptosis, and angiogenesis events.

(A) IHC staining of tumor tissues at 13 weeks showing a decrease in differentiation factor Ki67 (top), angiogenesis factor VIII (middle), and cyclin D1 (bottom) with degree of Pthrp ablation. (B) Western blot illustrating no change in PTH/PTHrP receptor 1 expression, but decreases in factor VIII, cyclin D1, and Bcl-2 with degree of Pthrp ablation. (C) Confocal images of IF staining in cultured cells isolated from tumors showing colocalization of PTHrP and cyclin D1 expression. The residual cells that escaped ablation and are still capable of expressing PTHrP are the only ones expressing cyclin D1 (homozygous, bottom row, arrowheads). Shown are DAPI (blue), PTHrP (red), and cyclin D1 (green). (D) TUNEL staining of breast tumor tissue (top) or in cells isolated from tumors and cultured (bottom), showing more abundant apoptotic events in homozygous tumors. Bottom panel: histogram showing average number of apoptotic cells per field in isolated tumor cells. Scale bars: 50 μm. Values represent mean ± SD. **P < 0.01.

Figure 6

Figure 6. PTHrP is involved in CXCR4 and AKT expression control.

(A) Western blot showing decreased CXCR4 expression in homozygous tumors. (B) Confocal images of IF. Top: primary breast tumors (control 13 weeks, homozygous mice 18 weeks). Bottom: cells isolated from these tumors and cultured. CXCR4 expression is significantly reduced with Pthrp ablation. Residual cells that escaped Pthrp ablation and are still expressing PTHrP are the only ones expressing CXCR4 (arrowheads). (C) IHC (left) and IF (right) images for AKT1 and AKT2 in tumors from control and ablated mice. Shown are DAPI (blue), AKT1 (top, red), and AKT2 (bottom, red). (D) Western blot showing decrease in AKT1 and increase in AKT2 concurrent with Pthrp ablation. (E) Confocal images of IF staining of cultured cells from control (top) and homozygous (bottom) tumors. The residual cells that escaped ablation and are still expressing PTHrP also express AKT1, although a small level of AKT1 is detectable in PTHrP-negative cells. Shown are DAPI (blue), PTHrP (green), and AKT1 (red). (F) Western blot of tumor extracts for AKT1 Ser473 phosphorylation. Scale bars: 50 μm.

Figure 7

Figure 7. AKT1 inhibition by siRNA enhances Pthrp ablation effect on tumor cell growth inhibition.

(A) Western blots for AKT1 expression in control (top) and homozygous cells (bottom) transfected with AKT1 siRNA or mock sequence (48 or 72 hours). (B) Proliferation of isolated Pthrpflox/flox tumor cells from control (Cre–) or homozygous (Cre+) mice after AKT1 siRNA or mock transfection (72 hours). Representative experiment out of 3 replicates. **P < 0.05 for all except Cre– mock versus Cre+ siRNA: P < 0.0001. Error bars represent SD.

Figure 8

Figure 8. PTHrP drives metastatic spread.

(A) Matrigel growth of tumor cells from Pthrpflox/flox;Cre– or Pthrpflox/flox;Cre+ tumors and histogram showing reduced invasive capacity for ablated cells (22 hours). **P < 0.01. (B) Cell motility test after wounding (48 hours); _Pthrp_-ablated cells show slower motility than control cells. (C) Epimet stain of cytospins for detection of circulating tumor cells (arrowheads show pan-cytokeratin–positive cells, 18 weeks). No tumor cells are detectable in blood of homozygous mice. (D) Tumor cells flushed from bone marrow (IF stain: cytokeratin 8/PyMT double positives) in control animals only. (E) Lung metastases are slower to appear in heterozygous mice. (F and G) Lung metastases appear in homozygous mice even later (between 13 and 18 weeks). (H and I) Lung metastases after MFP injection of adenovirus-transfected tumor cells. Lung metastases appear earlier in control mice than in the spontaneous tumor model (E and G), but are not detectable in homozygous ablated mice (adenovirus transfected) at 16 weeks. (All groups for E to I, n = 9). Scale bars: 100 μm (A–C); 50 μm (D); 200 μm (E, F, and H). Error bars represent SD. Large single and double asterisks refer to corresponding stages in Figure 9.

Figure 9

Figure 9. Spontaneous lung metastases are PTHrP and CXCR4 positive.

H&E stain (left) and IF confocal (right) of spontaneous lung metastases (no adenovirus) at same-size tumor (control 13 weeks, homozygous 18 weeks); lung metastases in homozygous mice are PTHrP and CXCR4 positive. Shown are DAPI (blue), CXCR4 (green), and PTHrP (red). Large single and double asterisks indicate corresponding stages illustrated in Figure 8E. Scale bars: 50 μm.

Figure 10

Figure 10. Anti-PTHrP neutralizing mAbs inhibit breast cancer progression in vitro and in vivo.

(A) Proliferation in Matrigel (24 hours) of human MDA-MB-435 breast cancer cells showing growth-inhibition effect of neutralizing antibodies 158 and M45 in vitro.*P < 0.05. (B) Tumor volume over time after injection of MDA-MB-435 cells into the MFPs of BALB/c nu/nu mice and treatment with anti-PTHrP mAbs, showing the tumor-reducing effect in vivo. Data are expressed as means of 8 mice in each group. *P < 0.05; **P < 0.01. (C) IF confocal images of mammary tumors 6 weeks after injection of MDA-MB-435 cells in MFPs of nude mice showing decrease of CXCR4 (top panels) and AKT1 (bottom panels) in treated animals. Shown are DAPI (blue), CXCR4 (green), and AKT1 (red). (D) H&E staining of lung metastases 6 weeks after injection of MDA-MB-435 in MFPs. Treatment with anti-PTHrP mAbs reduces the size and numbers of lung metastases. (E–G) Fewer mice present lung metastases after treatment with either mAb, and the metastases are smaller and fewer in numbers in treated animals. Mean ± SEM. *P < 0.05 (E); *P = 0.013 (F); *P = 0.045 (G). (H) IF confocal images of lung metastases in nude mice injected with MDA-MB-435 cells treated (6 weeks) or not with anti-PTHrP mAbs. Lung metastases are CXCR4 positive irrespective of treatment. Shown are DAPI (blue) and CXCR4 (green). Scale bars: 100 μm (A); 50 μm (C and H); 200 μm (D).

Figure 11

Figure 11. PTHrP influences several key steps in breast cancer.

Interactions are described here for PTHrP in tumor cell proliferation; through its effects on cell proliferation factor Ki67, cell-cycle progression regulator cyclin D1, and the G0/G1 to S transition, PTHrP is involved in very early steps of oncogenesis. PTHrP influences breast tumor cell survival, apoptosis, and angiogenesis through control of levels of expression for crucial signaling molecules such as AKT1/AKT2, Bcl-2, and factor VIII. Of great interest is the observation that PTHrP is involved in the control of CXCR4 expression and consequently also plays a role in metastatic spread.

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