Lung carcinoma progression and survival versus amino- and carboxyl-parathyroid hormone-related protein expression

Abstract

Purpose: Expression of the carboxyl PTHrP region of parathyroid hormone-related protein (PTHrP) is a positive prognostic indicator in women with lung cancer, but amino PTHrP is a negative indicator in other lung cancer patients. This project investigated whether PTHrP could be expressed as predominantly amino PTHrP or carboxyl PTHrP in individual lung carcinomas. It also assessed domain-specific effects on cancer progression and patient survival.

Methods: PTHrP immunoreactivities were analyzed versus survival in a human lung cancer tissue microarray (TMA). Growth was compared in athymic mice for isogenic lung carcinoma xenografts differing in expression of amino and carboxyl PTHrP domains.

Results: In the TMA, 33 of 99 patient tumors expressed only one PTHrP domain, while 54 expressed both. By Cox regression, the hazard ratio for cancer-specific mortality (95% confidence interval) was 2.6 (1.28-5.44) for amino PTHrP (P = 0.008) and 0.6 (0-2.58) for carboxyl PTHrP (P = 0.092). Xenografts of H358 lung adenocarcinoma cells that overexpressed amino PTHrP grew twice as fast as isogenic low PTHrP tumors in athymic mice, but growth of tumors expressing amino plus carboxyl PTHrP was not significantly different than growth of the control tumors. In summary, the presence of amino PTHrP signifies worse prognosis in lung cancer patients. In mouse xenografts, this effect was abrogated if carboxyl PTHrP was also present.

Conclusion: Amino PTHrP and carboxyl PTHrP can vary independently in different lung carcinomas. Carboxyl PTHrP may temper the stimulatory effect of amino PTHrP on cancer progression.

Keywords: Biochemical markers; Carcinoma, non-small cell lung; Molecular diagnosis and prognosis; Neuroendocrine and other endocrine factors; Oncogenes; Tumor suppressor proteins.

Fig. 1

Histograms of H-scores for PTHrP immunoreactivity in TMA lung cancer specimens. The upper panels show univariate histograms for amino PTHrP staining on the left and carboxyl PTHrP on the right. Both histograms include a preponderance of tumors scoring 0 and broad distribution of tumors with scores above 50. Thus, low staining was defined as a score of 50 or below for each PTHrP epitope. The bottom graph shows amino PTHrP and carboxyl PTHrP staining for patients divided on the basis of high and low tumor staining for each epitope. The largest group had significant staining for both domains, but tumors in some patients were restricted to amino PTHrP or carboxyl PTHrP immunoreactivity alone

Fig. 2

Amino PTHrP and carboxyl PTHrP expression in H358 cell lines transduced with lentiviral vectors. Conditioned media and underlying cell layer were harvested after 24 h in culture to assay PTHrP secreted into the media or remaining inside the cell. PTHrP values for each well were normalized to the total protein content of the cell layer within the well. The vector control line, transduced with empty lentiviral vector, expressed negligible levels of each domain. Amino PTHrP was increased compared to vector only in the PTHrP 1–87 and PTHrP 1–139 groups, as expected. Similarly, carboxyl PTHrP increased only in the PTHrP 1–139 and PTHrP 33–139 groups. Twenty-four hour secretion of PTHrP exceeded the quantity detected in the cells by a large margin for both domains. Data represent triplicate measurements ± standard error of the mean

Fig. 3

Time course of tumor growth for each PTHrP isoform group. The upper row of graphs shows tumor volume (estimated from caliper dimensions) plotted versus time for individual mice in each of the groups. Line segments connect the tumor volumes for each individual mouse from 1 week to the next. The greatest tumor volumes were observed for mice in the PTHrP 1–87 group. On average (lower graph), the PTHrP 1–87 group had significantly higher volumes than did the vector control group at week 4 (P < 0.05) and week 5 (P < 0.01). Three mice in the PTHrP 1–139 group died by week 4

Fig. 4

Effect of PTHrP isoform on lung tumor proliferation and angiogenesis markers. Graphs show counts of tumor mitotic bodies/50 high-power fields (left panel) and a staining index, % of area with marker immunofluorescence, for CD31, an endothelial marker, for mice bearing control tumors with low PTHrP (n = 6), and tumors overexpressing PTHrP 1–87 (n = 9), PTHrP 33–139 (n = 7), and PTHrP 1–139 (n = 6). PTHrP 1–87 expressing tumors had higher numbers of mitotic bodies than control tumors (P = 0.001), PTHrP 33–139-positive tumors (P = 0.001), and PTHrP 1–139-positive tumors (P = 0.109, non-significant). PTHrP 1–87 expressing tumors also had a greater CD31 staining index than control tumors (P = 0.001), PTHrP 33–139-positive tumors (P = 0.001), and PTHrP 1–139-positive tumors (P = 0.065, non-significant)

Fig. 5

Mouse weights over time based on tumor PTHrP expression. Mouse weight increased progressively with time over the 5 week after tumor implantation for the vector control mice. Growth slowed after 3 week for the PTHrP 1–87 and PTHrP 1–139 mice, and weights were 2–3 g less at week 4 and week 5 for mice in those groups on average than for mice in the control group (P < 0.01 for vector versus PTHrP 1–87 group)