Date of Award


Degree Type


Degree Name

Master of Science




Dr. Ann Chambers

Second Advisor

Dr. Alan Tuck


The integrin-binding protein osteopontin (OPN) has been associated with poor prognosis of breast cancer, and has been found to influence malignant properties of breast cancer cells, including adhesion, migration, invasion, and metastasis. OPN and the α9βl integrin have been implicated in many overlapping functions important to metastasis, including cell adhesion and migration, as well as the processes of angiogenesis and lymphangiogenesis. Our laboratory has previously derived a cell line (“468LN”) from weakly metastatic MDA-MB-468 (“468”) human breast cancer cells. This cell line has a drastically enhanced ability to metastasize to the lymph nodes, and has been shown to have up-regulated OPN and α9βl expression. To study the specific roles of OPN and α9βl in breast cancer metastasis, we have transfected 468 cells to overexpress OPN and α9, alone (468-CON-OPN and 468-CON-α9, respectively) or in combination (468-OPN- α9), and assessed functional differences in malignant behavior in vitro (cell proliferation, adhesion, and migration) and in vivo (tumorigenicity and spontaneous metastasis following mammary fat pat injection into nude mice). In vitro assays indicate that either OPN or α9βl expression (alone or in combination) did not affect cell proliferation doubling time. OPN stimulated adhesion and migration of mock-transfected 468 (468- CON-CON) (adhesion: P=0.0084; migration: P<0.0001) and α9-transfected 468 (468- CON-α9) (adhesion: P=0.0002, migration: P<0.0001) cells, compared to PBS negative control. Interestingly, 468-CON-α9 had a 70% higher adhesive response and a 5-fold higher migratory response to OPN compared to 468-CON-CON (adhesion: P=0.0003; migration: P<0.0001). Antibodies to α9βl successfully inhibited the enhanced adhesive response to OPN observed both in 468-CON-CON (48% reduction; P=0.0175), and at a iii much higher degree in 468-CON-α9 cells (114% reduction; P<0.0001), compared to IgG negative control. Taken together, these results suggest that the interaction between α9βl and OPN may be important for mediating adhesion and migration in 468 cells. 468- CON-CON, 468-CON-α9, 468-OPN-CON, and 468-OPN-α9 were inoculated into the mammary fat pad of athymic nude mice (xenograph model) to assess the molecular mechanisms by which OPN and α9βl influence the tumorigenicity and metastatic abilities of these cell lines. The expression of α9βl in 468-CON-a9 and 468-OPN-a9 increased the lag time of primary tumor growth, resulting in the development of primary tumors with 2.4-fold smaller average tumor volumes as compared with 468-CON-CON (P<0.001 for both 468-CON-α9 and 468-OPN-α9). Although the overexpression of OPN in 468-OPN-CON increased the incidence of lymph node metastasis as compared with 468-CON-CON (P=0.0211), the overexpression of α9βl in 468-CON-a9 did not affect lymph node metastasis, suggesting that OPN-mediated lymph node metastasis may involve the interplay of one or more of OPN receptors. This work has demonstrated an important role of OPN-α9βl signaling in breast cancer malignancy in vitro, but more work is required to assess its significance in vivo.



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