Polyamines and Breast Cancer Progression and Metastasis

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The natural history of human breast cancer is characterized by an inevitable transition from a relatively indolent hormone-dependent phenotype to a more aggressive typically hormone-independent status that is ultimately responsible for the patient's demise. Evidence indicates that activation of the polyamine pathway may be one of the contributing factors to breast cancer progression. Using an experimental system of mammary tumor progression, we found that an increase in cellular ODC activity was associated with transition to hormone independence and to a less differentiated and more metastatic phenotype (40). With respect to human breast cancer, increased ODC

Fig. 1. Effect of SAM486A on tumor growth (A) and polyamine (PA) levels (B) in nude mice. A total of 5 x 106 cells suspended in Hank's balanced salt solution were implanted ortho-topically into two axillary mammary fat pads per mouse of 4-wk-old female athymic nude mice. Two weeks later, when tumor areas averaged 80 mm2, mice were randomly assigned to receive increasing doses of SAM486A (0, 1 mg/kg, 5 mg/kg, or 10 mg/kg) daily, by ip injection. Tumor growth was monitored once per week by measurement of tumor area (length x width) and expressed as fold increase over baseline (A). After 3 wk of SAM486A administration, the mice were sacrificed, the tumors were removed and PA levels in the tumors were measured (B). Statistical analysis: (A) *p < 0.01 vs control (analysis of variance followed by Dunnett's multiple comparison test). (B) *p< 0.01 vs control. (Reproduced with permission from ref. 35.)

Fig. 1. Effect of SAM486A on tumor growth (A) and polyamine (PA) levels (B) in nude mice. A total of 5 x 106 cells suspended in Hank's balanced salt solution were implanted ortho-topically into two axillary mammary fat pads per mouse of 4-wk-old female athymic nude mice. Two weeks later, when tumor areas averaged 80 mm2, mice were randomly assigned to receive increasing doses of SAM486A (0, 1 mg/kg, 5 mg/kg, or 10 mg/kg) daily, by ip injection. Tumor growth was monitored once per week by measurement of tumor area (length x width) and expressed as fold increase over baseline (A). After 3 wk of SAM486A administration, the mice were sacrificed, the tumors were removed and PA levels in the tumors were measured (B). Statistical analysis: (A) *p < 0.01 vs control (analysis of variance followed by Dunnett's multiple comparison test). (B) *p< 0.01 vs control. (Reproduced with permission from ref. 35.)

activity has been shown to be associated with adverse prognostic features such as high cellularity, low histological differentiation, high nuclear aplasia, peritumoral lymphatic and blood vessel invasion, high S-phase fraction, and cathepsin-D expression (11,41).

In a pilot study involving a cohort of 50 primary human breast cancers, we have observed that ODC activity was a strong negative, independent prognostic factor of overall survival 42). Figure 2 illustrates the effect of a 10-fold difference in ODC activity on overall survival after adjustment for other prognostic factors. This observation has been subsequently confirmed in a larger cohort of patients (11). Because patients' survival is strictly related to the development of metastasis, this finding strongly suggests that increased ODC activity and hence, activation of polyamine biosynthesis, is involved in the metastatic process.

To address the involvement of polyamines in breast cancer invasiveness and metastasis, we turned to the hormone-independent MDA-MB-435 and MDA-MB-231

Time (years)

Fig. 2. Influence of a 10-fold difference in ODC (_, ODC = 10 pmol/mg;----ODC =

100 pmol/mg) on breast cancer survival after adjustments were introduced for estrogen receptor and lymph nodal status. (Reproduced with permission from ref. 42.)

Time (years)

Fig. 2. Influence of a 10-fold difference in ODC (_, ODC = 10 pmol/mg;----ODC =

100 pmol/mg) on breast cancer survival after adjustments were introduced for estrogen receptor and lymph nodal status. (Reproduced with permission from ref. 42.)

human breast cancer cell lines which manifest an aggressive behavior both in vitro and in vivo. We found that DFMO administration, in addition to inhibiting cell proliferation, reduced invasiveness in matrigel of both cell lines by 70% (43). Furthermore, DFMO treatment inhibited the ability of MDA-MB-435 cells to grow in a "branching" pattern in the matrigel outgrowth assay, which is a feature of aggressive tumors. More importantly, DFMO treatment significantly inhibited the development of pulmonary metastasis from MDA-MB-435 human breast cancer cells orthotopically implanted into the mammary fat pad of athymic nude mice (43).

Using the same orthotopic model system, we next tested whether DFMO inhibits lung colonization by tumor cells or expansion of single-cell deposits into metastasis (44). To address this issue, we took advantage of the availability of green fluorescent protein-tagged MDA-MB-435 cells, which can be tracked at the single-cell level throughout the entire lung. As shown in Table 1, DFMO treatment was again found to be effective in suppressing pulmonary metastasis. DFMO reduced the development of both multicellular and single-cell metastatic deposits, thus suggesting that its effect was primarily preventing lung colonization, rather than halting progression of single-cell deposits to overt metastasis. If the latter were true, one would have anticipated a higher single cell to multicellular metastasis ratio in the DFMO-treated mice. The findings reported in Table 1 also indicate that a more prolonged duration of treatment did not result in a superior antimetastatic effect, whereas discontinuation of DFMO was not associated with an increase in pulmonary metastasis, at least during the 4-wk period of observation. It is worth emphasizing that in this experimental system, the antimetastatic effect of DFMO was totally independent of any suppressive effect on the growth of the primary tumor. As a matter of fact, as shown in Fig. 3, the growth of the primary tumor was not affected at all by DFMO treatment, despite a complete suppression of putrescine and a 70% decrease in cellular levels of spermidine induced by DFMO.

To test the general applicability of these observations, in collaboration with Dr. Dan Welch at the University of Alabama Cancer Center, we are testing the effect of DFMO in

Table 1

Effects of the Different DFMO Treatment Regimens on Pulmonary, Lymph Nodal Metastasis, and Local Recurrence from GFP-Tagged MDA-MB-435 Breast Cancer Xenografts in Nude Mice

Pulmonary metastasis

Table 1

Effects of the Different DFMO Treatment Regimens on Pulmonary, Lymph Nodal Metastasis, and Local Recurrence from GFP-Tagged MDA-MB-435 Breast Cancer Xenografts in Nude Mice

Pulmonary metastasis

Experimental Groups

No. of mice with metastasis

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