Molecular Cancer Biology Program
PO Box 63 (Haartmaninkatu 8)
FI-00014 University of Helsinki
Tel. +358 9 191 25537
Fax. +358 9 191 25610
A hallmark of the malignant process is the acquisition of an invasive phenotype that allows neoplastic cells to invade the surrounding tissue and disseminate into specific organs. Metastatic spread of cancer is the most serious challenge for cancer treatment and the metastatic dissemination, rather than the primary tumor, is responsible for 90% of cancer deaths. Cancer metastasis is a multistep process the details of which are still poorly understood. Cancer cell must detach from the primary tumor, enter the vasculature (intravasation) to be transported to distant organs where it must arrest, extravasate into the parenchyma, survive, and proliferate to form a metastatic lesion. Recently, it was suggested that this complex metastatic cascade could be simplified into two major phases: i) physical translocation from the primary tumor to the distant organ and ii) colonization at the distant site [Chaffer, 2011 #1]. In addition, cells can stay dormant (no proliferation or apoptosis) at the secondary sites and metastases can occur after long latency periods that range from years to decades after the primary treatment [Goss, 2010 #31].
We aim at identifying novel tumor progression/invasion associated molecules using peptide and antibody phage display technology and comparative proteomics with the aid of mass spectrometry from two very aggressive cancer models: i) human glioblastoma models with differential invasion potential and ii) cultured metastatic vs. non-metastatic human melanoma/breast cancer cells. The functional role of the identified molecules will be studied in 2D and 3D cell culture models as well as in tumor progression and its metastatic spread using preclinical animal models of human cancer. In addition, we study the expression of the identified cancer-associated molecules in various human tumors, and evaluate their association with clinicopathological variables and patient survival in collaboration with the clinicians. Furthermore, we will use peptide, virus or antibody-targeted therapy to prevent tumor growth and its metastatic spread.
We use two different human carcinoma cell lines as a model for the metastatic spread of cancer. These cell lines were derived by serial dilution cloning from the MDA-MB-435 carcinoma cells followed by screening for the metastatic potential in athymic mice [Urquidi, 2002 #39]. This cell line was originally considered as breast carcinoma, but microarray data suggests that it may be of melanoma origin [Ellison, 2002 #46;Rae, 2007 #47;Ross, 2000 #52]. Recent reports claim again that the cell line would be a breast carcinoma [Chambers, 2009 #91;Hollestelle, 2009 #92]. However, the common origin of these cell lines enables the comparative investigation of cellular and molecular events in the metastatic process in a stable and isogeneic model [Urquidi, 2002 #39]. Both the metastatic and non-metastatic cells are able to reach the lungs of tumor-bearing mice, while only the metastatic cells can colonize the lung and form metastatic lesions. The non-metastatic cells survived but stayed dormant since tumorigenic cells could be retrieved from the lungs [Goodison, 2003 #40]. Thus, this cell model mimics the second phase of metastasis, colonization at the distant site.
Using the 2D gel analysis we recently identified nucleophosmin (NPM) to be overexpressed in the non-metastatic cells compared to the metastatic cells. We studied the role of NPM further in cell culture models of breast cancer and in a large array of human breast carcinoma samples (n = 1160). We showed that reduced levels of NPM protein were associated with poor prognosis of patients. Furthermore, luminal epithelial cells of the histologically normal breast displayed high levels of NPM and overexpression of NPM in the invasive MDA-MB-231 cells abrogated their growth in soft agar. These results support a tumor suppressive role for NPM in breast cancer [Karhemo, 2011 #11].
Due to the hydrophobic and complex nature of the cell surface proteins we have performed a comparative and quantitative, non-gel based mass spectrometric cell surface proteome analysis of our metastatic and non-metastatic cell lines using cell surface biotinylation and streptavidin pull down followed by trypsin digestion and LC-MS/MS.
According to the quantitative analysis 23 of the identified proteins showed statistically significant difference in their expression between the cell lines varying from 1.3-fold to 8.8-fold (Anova <0.05). We selected the 2-fold expression difference as a threshold for the further analyses. Sixteen proteins showed ≥ 2-fold expression differences, and interestingly, out of these 15 (94%) were overexpressed in the metastatic cells. About 1/3 of these proteins have been previously associated with metastasis. However, 2/3 of the identified proteins were either unknown or known proteins that have not previously been associated with metastatic spread of cancer. Up to date we have validated the differential expression of five proteins. Currently, we are validating their functional role in the metastasis spread of cancer using 2D and 3D cell culture models as well as preclinical models of human cancer.
Figure 1. Comparative proteomics. We isolated biotinylated cell surface proteins from an isogenic pair of metastatic and non-metastatic cells using a cleavable, cell impermeable biotin and magnetic streptavidin beads. Identification of the differential expression of the proteins was performed using the Progenesis soft ware. In addition, we analyzed how the identified proteins interacted with each other, and which signaling pathways they were involved in using the computational platform Moksiskaan [Laakso, 2010 #25].
Astrocytes arise from multipotent neural stem cells and retain their capacity for division throughout their life span. Low-grade astrocytomas, a class of malignant brain tumors, acquire their blood supply by propagating along the existing normal blood vessels in a process termed vessel co-option. This leads to diffuse invasion of tumor cells over long distances in the brain without formation of real tumor masses. The most malignant forms of astrocytomas, also called glioblastoma multiforme (GBM), become highly vascularized and tumors appear more local than the low-grade astrocytomas. Prognosis for patients suffering from malignant brain tumors is poor. Especially low-grade astrocytomas are challenging due to their diffuse growth pattern with invasion into normal brain, which make them incurable by conventional therapies such as radiation or surgery.
We have used in vivo phage display technology to identify a peptide that specifically homes to the diffuse astrocytoma islets harboring co-opted tumor vessels. In addition to this murine model of early stage astrocytoma the peptide also homes to the orthotopic U87MG human glioma xenografts. We have named our peptide “CooP”. We have also identified the receptor our peptide binds to in the brain tumors. Interestingly, the receptor is expressed in a grade dependent manner in human brain tumors. The more aggressive the tumor was the more expression of the receptor was detected. Overexpression of the receptor appears to promote invasion of the intracranial brain tumors. We are currently studying the mechanisms underlying this change in the invasive properties caused by the receptor expression.
Targeted delivery of nanoparticles
We are also designing novel nanoparticles decorated with our CooP peptide for the targeted delivery of drugs and siRNAs. Cell culture studies show that the peptide-containing nanoparticles bind better to the brain tumor cells expressing the receptor than to the parental cells. The in vivo delivery of these particles to the intracranial tumors is ongoing. If the particles home to the tumor site also in vivo we will fill the particles with drug molecules or siRNAs to study if we can treat these very malignant tumors in the preclinical models of human gliomas.
March 20, 2012
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