Neoplastic cells growing under hypoxic conditions exhibit a more aggressive phenotype by activating a cascade of molecular events partly mediated by hypoxia-inducible transcription factor (HIF-1α) and vascular endothelial growth factor (VEGF). We therefore hypothesized markers of hypoxia would correlate with the prognosis of osteosarcoma in part through defining chemotherapy response and in part through defining the metastatic phenotype. Thus, we determined the frequency of HIF-1α expression, VEGF expression, degree of vascularization, and cell proliferation in osteosarcoma patients as markers of hypoxia and determined preliminarily if these measures predicted the clinical outcome through correlation with chemotherapy response and metastases.
The major limitation of our study is that it is an immunohistochemical analysis of an existing patient cohort. Although this group represents all material available from a single large institution, in any analysis of this type, the results should be considered exploratory. Any associations identified may not necessarily be causal. One must be cautious in interpreting data. In particular, our primary clinical parameters being assessed were Huvos grade and metastases (outcome), and the relationship of VEGF positivity with gender was an unexpected finding. Although potentially justifiable scientifically as discussed later, confirmation in a subsequent study will be crucial. Finally, limited sample size severely curtails the power of the study. Negative results cannot be interpreted as a nonexistent association, and the ability to analyze subgroups was limited. Despite these limitations, interesting results were obtained. Validation of these results in a subsequent prospective patient cohort has the potential to identify predictive and prognostic factors supporting the utility of reporting these results.
Our results expand upon previous work regarding hypoxia markers and osteosarcoma, and demonstrate an association between HIF-1α expression and metastatic disease. In 61% of HIF-1α-positive patients, metastases were present, representing a 4.3-fold greater risk for having metastatic disease in patients with HIF-1α expression compared to that of HIF-1α-negative patients. In addition, increased VEGF protein expression in osteosarcoma samples from female patients versus male patients was seen. Finally, we noted an insignificant trend for higher frequency of VEGF expression to be present in the high-grade as compared to low-grade osteosarcoma. Further analysis of larger cohorts of low-grade osteosarcoma samples would be worthwhile in order to confirm or refute the trend.
Hypoxia induces the expression of a number of hypoxia-regulated genes. Induction of HIF-1α protein appears to be a critical step in the hypoxic response and occurs via increased mRNA expression, protein stabilization, nuclear translocation, and augmented activity of its transcriptional activation domains [37
]. Zhong et al. [50
] reported most normal human tissues showed no HIF-1α immunoreactivity (153 of 174 clinical specimens [88%]). Furthermore, they reported overexpression of HIF-1α protein in 69 of 131 primary malignant tumors (53%), representing 13 of 19 tumor types screened [50
]. Recent studies using the human osteosarcoma cell line SaOS-2 demonstrated a relationship between hypoxia and increased HIF-1α protein expression and subsequent VEGF expression [47
]. Our data suggest this increased HIF-1α expression is an important marker for metastases in osteosarcoma patients. Metastases were present in 61% of the HIF-1α-positive patients, and 73% of HIF-1α-negative patients showed no evidence of metastases. These results for osteosarcoma correlate with studies reporting a higher incidence of overexpression of HIF-1α in metastases [4
]. We found no other association between HIF-1α protein expression and clinical data.
Wu et al. [47
] reported evidence of increased VEGF expression in a human osteosarcoma cell line. We observed VEGF protein expression in 73% of patients. More specifically, an association between VEGF expression and female gender was demonstrated. The mechanism for this relationship is not entirely clear. A relationship between estrogen and bone tissue is well-known, mainly through clinical studies where 17β-estradiol (E2) deficiency in postmenopausal or ovariectomized women caused a rapid loss of trabecular bone. Furthermore, in both men and women, estrogen deficiency is associated with an age-related sustained bone loss that can lead to osteoporosis [25
]. Although the association between gender and VEGF expression has not been clearly determined, in classic hormone-dependent tissues such as the endometrium, VEGF gene transcription is increased under the influence of estrogen [27
]. Koos et al. [21
] observed estrogen induces rapid, transient binding of HIF-1 to the promoter, which subsequently mediates VEGF transcription in response to hypoxia. One study, however, reported no relationship between VEGF expression and gender [51
], suggesting differential VEGF expression in various cancers.
Correlations between VEGF expression and increased vascularization have already been demonstrated in osteosarcoma. Using biopsies from 30 osteosarcoma patients, Lee et al. [24
] detected VEGF mRNA transcripts in all primary osteosarcoma samples and nine xenograft specimens examined and showed the number of vessels in VEGF-positive tumors was greater than in VEGF-negative tumors. Kaya et al. [18
] studied 27 primary osteosarcoma samples and found VEGF was positive in 63% of samples. Microvascular density was higher in VEGF-positive tumors than in VEGF-negative tumors [18
]. Other investigators have suggested VEGF expression does not always relate to increased tumor neoangiogenesis. In non-small-cell lung cancer, about ½ of the cases expressing VEGF had poor vascular density, which suggested VEGF-induced angiogenesis may be dependent on other regulators [10
]. We observed no relationship between VEGF expression and the microvascular density in our patient samples.
Malignant progression is another hallmark of the poor outcome of solid tumors. In giant cell tumor of bone, overexpression of VEGF is associated with the biologic behavior of the tumor as determined by clinical parameters. Stage III giant cell tumors display relatively higher levels of VEGF gene expression than Stage I/II tumors [49
The final marker we investigated on osteosarcomas was the related cell proliferation utilizing a Ki-67 index. Ki-67 is a nuclear antigen that is only expressed in proliferating cells and is often used as an estimate of tumor growth. Studies on breast cancer have demonstrated high concentrations of HIF-1α were associated with increased proliferation as shown by positive correlations with Ki-67 [2
]. Analysis of 25 head and neck osteosarcomas demonstrated 88% were positive for Ki-67 [17
]. In a different study, the positive rate of Ki-67 in recurrent osteosarcoma was 81.8% but only 35.5% in the primary tumor [30
]. In addition, Ki-67 positivity is believed one of the risk factors for recurrence in meningiomas. However we observed no association between the Ki-67 labeling index and clinical data. The high proliferative potential of tumor cells as a prognostic factor has given variable results. Some studies described a more favorable outcome for tumors with higher labeling indices [22
]. However, proliferation indices were not correlated with overall outcome for patients with rectal or cervical cancers [5
We examined hypoxia markers in osteosarcoma. HIF-1α is overexpressed in a substantial proportion of patients with metastases, confirming previous work showing this relationship in several other cancers. Previous studies have suggested HIF-1 mediates resistance to chemotherapy and radiation [15
]. Targeting the HIF-1 pathway, therefore, has become an important area for cancer therapy research. Tan et al. [39
] demonstrated inhibition of HIF-1α protein synthesis in several cancer lines including human breast cancer, prostate cancer, and glioblastoma prevented the activation of HIF-1 target genes, such as VEGF and glucose transporter-1. Using small hairpin RNAs to target the expression of the HIF-1α gene in human osteosarcoma cell line SaOS-2, Wu et al. [48
] blocked the hypoxia transduction pathway efficiently and inhibited the growth of osteosarcoma cells; expression of the HIF-1α gene was inhibited by 90%. This direction of research shows promise for the development of even more efficient cancer therapies for osteosarcoma. We also observed an overexpression of VEGF protein in female patients, which may support previous work postulating VEGF gene transcription is increased under the influence of estrogen. Studies investigating breast cancer are currently considering cyclooxygenase-2 (COX-2) inhibitors as a possible mechanism for cancer therapy. Substantial evidence exists linking COX-2 to the development of cancer [38
]. Furthermore, COX-2 expression in ductal carcinoma in situ has been correlated with VEGF and HER-2/neu [31
]. Although COX-2 has no relationship to estrogen receptors [7
], further investigation should reveal the role estrogen plays in the development of cancer. Finally, we also observed a trend describing a relationship between VEGF expression and tumor grade, an area that needs further examination to prove its importance in osteosarcoma and patient prognosis. The data provides novel findings regarding hypoxic markers in human osteosarcoma and introduces new areas for further investigation in osteosarcoma research.