**4. Conclusions and perspectives**

56 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

Expression of IGFBP7 in lung cancer cell lines using RT-PCR revealed decreased expression of IGFBP7 compared to controls, and 42 out of 90 patients with primary lung tumors exhibited negative staining of IGFBP7 by immunohistochemical analysis [118]. There was a significant correlation between DNA methylation of exon/intron 1 region and IGFBP7 downregulation. When a p53 expression vector was transfected into lung cancer cell lines, it could only induce expression of IGFBP7 in the unmethylated cell line, but not in the methylated cell lines, suggesting that IGFBP7 might be regulated by p53 in lung cancer cell

A study found that a single nucleotide polymorphism (G to A) in the IGFBP7 promoter region was significantly associated with a reduced risk of SCCHN, when analyzed in a hospital-based case-control study of 1065 SCCHN patients and 1112 cancer-free control subjects. Upon analyzing reporter gene constructs, the G to A allelic change at -418 of the IGFBP7 promoter had increased promoter and DNA binding activity, suggesting increased

Although IGFBP7 has been shown to function as a tumor suppressor in a wide variety of cancers, a few studies suggest that IGFBP7 has an opposite effect, ie. promoting cancer growth. These cancers include the blood cancer, leukemia, and the brain cancer,

IGFBP7 is a selective biomarker of glioblastoma (GBM) vessels, strongly expressed in tumor endothelial cells and vascular basement membrane [120]. IGFBP7 was strongly expressed in GBM specimens but not nontumor brain tissue. Moreover, statistical analysis showed that expression of IGFBP7 correlated inversely with overall GBM survival rates. Inhibition of IGFBP7 expression using siRNA transfection in a glioma cell line inhibited cell growth [121]. Addition of IGFBP7 to cell culture medium stimulated cell proliferation. IGFBP7 also promoted glioma cell migration, through downregulation of AKT phosphorylation and enhanced ERK1/2 activation [121]. IGFBP7 expression in brain endothelial cells was found to be upregulated by secreted factors from GBM cells through TGF-1/ALK5/Smad2 signaling pathway, which has been implicated in angiogenesis

Overexpression of the human gene BAALC (brain and acute leukemia, cytoplasmic), was shown to be associated with inferior outcome and chemotherapy resistance in adult patients with cytogenetically-normal acute myeloid leukemia (CN-AML), T cell-acute lymphoblastic leukemia (T-ALL) and B-precursor acute lymphoblastic leukemia (B-ALL)[123,124,125,126,127]. IGFBP7 was strongly correlated with BAALC-expression, implicating IGFBP7 in acute leukemia [128]. Aberrent expression of IGFBP7 in adult leukemia was correlated with chemotherapy resistance and inferior survival. Addition of IGFBP7 to leukemic cell lines inhibited cell growth without induction of apoptosis or senescence, suggesting a role of IGFBP7 in contributing to drug resistance through reduced sensitivity to cytostatic drugs [128]. Aberrently increased levels of IGFBP7 were found in

*Lung cancer* 

lines.

glioblastoma. *Glioblastoma* 

[122].

*Acute leukemia* 

*Squamous cell carcinoma of the head and neck (SCCHN)* 

IGFBP7 protein expression [119].

IGFBP7 has been shown to have tumor suppressive function in breast and other cancers. When examining the summarized data in Table 1, a common thread appears. Overexpression of IGFBP7 leads to inhibition of growth both *in vitro* and *in vivo*, increased expression of apoptotic markers (caspases, cleaved PARP), senescence associated proteins (*i.e.* p21, p27, p53), and decreased expression of proteins associated with proliferation (p-ERK). IGFBP7 appears to affect signaling through the MAP kinase pathway in many tumor models, including breast cancer. OIS may be a mechanism of tumor suppression by IGFBP7. The breast cancer cell lines used in our study, MDA-MB-468 cells, have a mutated PTEN, disregulating the PI3K pathway [131]. OIS can be triggered not only by the activation of oncogenes but also by the loss of tumor suppressor genes, such as PTEN. By upregulating proteins that counteract proliferation, such as cyclin dependent kinase inhibitors, *ie.* p21, which we have shown to occur upon IGFBP7 addition to breast cancer cells, the combined effect can lead to OIS [132]. Our model for the role of IGFBP7 in breast cancer inhibition depicts the entrance of IGFBP7 full length or cleaved IGFBP7 (through matriptase) into the cell, where signals are propagated to the nucleus, leading to the upregulation of expression of cyclin dependent kinase inhibitors, such as p21 and p27 (fig 7). This together with an already hyperstimulated MAP kinase pathway due to oncogenic mutations such as RAS, leads to MAP kinase pathway inhibition, growth arrest, and senescence, as suggested by the conflicting signal model of senescence[132].

The strong link to breast cancer outcome suggests that IGFBP7 may not only be a good prognostic indicator for malignant disease progression, but also a useful surrogate marker for monitoring therapeutic responses in the treatment of breast cancers. Senescence has been shown to be a method of halting tumor growth by many standard chemotherapeutic drugs [133]. Preliminary results indicate that senescence may be one mechanism by which IGFBP7 inhibits breast cancer cell growth in our system. Inhibition of breast cancer growth in vivo and in vitro together with induction of senescence indicates that IGFBP7 could be further developed as a potential drug to treat breast cancers. The fact that IGFBP7 has growth inhibitory effects when expressed in triple negative breast cancer cells, *i.e.* MDA-MB-468, provides an exciting opportunity to bring to the clinic a potential drug for hard to treat breast tumors.

Insulin-Like-Growth Factor-Binding-Protein 7: An Antagonist to Breast Cancer 59

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Fig. 7. Model for IGFBP7-mediated inhibition of breast cancer cell growth. IGFBP7 full length (FL) is cleaved by cell surface matriptase to short form (SF). Both forms enter breast cancer cells through an as yet unknown receptor, followed by signal propagation to the nucleus, which leads to upregulation of expression of cyclin dependent kinase (CDK) inhibitors, such as p21 and p27. This ultimately leads to growth arrest and senescence.
