An osteopontin splice variant induces anchorage independence in human breast cancer cells

Abstract

In malignant tumors, metastasis genes are typically deregulated by aberrant expression or splicing. Osteopontin is expressed at high levels by various cancers and contributes importantly to their invasive potential. In contrast, osteopontin derived from host cells induces cellular immunity and could bolster antitumor protection by cytotoxic T lymphocytes. Here we show that breast cancer cells express multiple splice variants of osteopontin. According to RT-PCR analysis of human breast tissue specimens, the splice variant osteopontin-c is a highly specific marker for transformed cells, which is not expressed in their surrounding normal tissue. The fulllength form of osteopontin aggregates in the presence of physiologic amounts of calcium and, in this state, leads to enhanced cell adhesion. Ostensibly, this effect is inhibitory for tumor cell dissemination. The shortest splice variant, osteopontin-c, does not aggregate in the presence of calcium and enhances clone formation in soft agar. According to microarray analysis, osteopontin-c induces the expression of oxidoreductases, consistent with protection from anoikis during anchorage-independent growth. These studies define a third functional domain of osteopontin, beside the C-terminal CD44-binding site and the central integrin-binding site. They also provide evidence for a bifunctional character of osteopontin, with the soluble form supporting invasiveness and the aggregated form promoting adhesion.

Osteopontin: Role in immune regulation and stress responses

“…Furthermore, OPN promotes proliferation and survival of IL-3-dependent bone marrow cells; anti-CD44 antibody attenuates these effects (Lin et al, 2000). A C-terminal location has also been suggested as the site of CD44 interaction (Weber et al, 2002;He et al, 2006). A monoclonal antibody recognizing the Cterminal region of OPN blocked the ability of cells to attach to OPN, supporting the possibility that attachment of OPN to CD44 modulates the cells' ability to bind to the RGD binding motif via the RGD sequence (Kazanecki et al, 2007a).…”

Metabolism in cancer metastasis

“…[25][26][27] Therefore, anchorage-independent survival may be more critical to the process of cancer metastasis than organ-specific homing. 28 Cancer cells that have been shed from a primary tumor need to overcome the energy deficit to survive and form metastases. Consistent with the requirements, increased cancer invasiveness under deadherent conditions is associated with higher mitochondrial activity, elevated ATP production, pyruvate uptake, and oxygen consumption.…”

“…By contrast, the full-length form osteopontin-a is present in both breast cancers and nontransformed breast tissue. 28,49 One role for osteopontin in tumor progression is the support of anchorage-independence. Osteopontin-c is never expressed without the full-length form osteopontin-a, and the two variants may synergize in supporting the survival of circulating tumor cells.…”

Influence of osteopontin expression on the metastatic growth of CC531 rat colorectal carcinoma cells in rat liver

“…11 Specific splice variants have been associated with their expression by breast cancer and are thought to function as supportive for neoplastic growth. 12,13 In line with this, Opn has been identified as lead marker of colon cancer progression, with the highest mRNA expression levels found in liver metastasis. 14 OPN has several domains including a conserved thrombin cleavage site (amino acids R168-S169 in human OPN), 8 which splits the protein into two functional fragments.…”

“…The existence of tumor-specific Opn forms has already been reported for breast cancer. 12,13 The assumption that the smaller Opn bands are instrumental for cancer growth is supported by the observation that a 28 kDa fragment of rat OPN, which resulted from the digestion of OPN by endoproteinase Arg-C, was active in cell attachment via a non-RGD sequence. 31 After 2 weeks, the treatment induced reduction in Opn expression corresponded to a 488% decreased tumor load in response to the maximum ASO Opn dosage.…”