Par-4 is a negative regulator of mammary tumor recurrence

Abstract

Abstract The natural history of breast cancer is frequently characterized by tumor relapse following initial treatment. Recurrent tumors, which can arise locally or at distant sites, are responsible for the majority of deaths from breast cancer. At present very little is known about what causes tumors to recur; consequently, a detailed understanding of the cellular and molecular mechanisms underlying tumor recurrence would contribute significantly to the treatment of breast cancer. To better understand the mechanisms of tumor recurrence, our laboratory has generated mouse models that faithfully recapitulate many key aspects of breast cancer recurrence in humans. These models employ the tetracycline-inducible system to allow for mammary-gland specific, doxycycline-inducible expression of various oncogenes, including Myc, Neu, and Wnt1. Oncogene expression consistently leads to formation of mammary gland tumors, and de-induction of the oncogene frequently leads to complete regression of the primary tumor. However, following a period of latency during which the tumors remain non-palpable, tumors frequently recur, and these recurrent tumors are no longer dependent upon the oncogene that initiated their growth. These models thus provide an ideal system in which to investigate the cellular and molecular mechanisms of breast cancer recurrence. To gain insight into these mechanisms, we first performed gene expression profiling to compare primary tumors initiated by Myc, Neu, and Wnt1 with their corresponding recurrent tumors. We identified a cohort of genes whose expression was significantly altered in recurrent tumors in all 3 models. Principal component analysis revealed that recurrent tumors arising in all 3 models had very similar transcriptional profiles, and these profiles differed substantially from the corresponding primary tumor. Thus these models share a common recurrence signature, suggesting that these tumors may employ a similar mechanism of recurrence. We next examined whether the genes comprising this signature are functionally involved in regulating recurrence. We focused our attention on one gene present in the recurrence signature, the tumor suppressor par-4. We found that par-4 expression is decreased in recurrent tumors arising from Neu-, Myc-, and Wnt-initiated primary tumors. To determine whether par-4 plays a causal role in regulating tumor recurrence, we suppressed its expression using virally delivered shRNA. Knockdown of par-4 in Neu-derived tumor cells led to a decrease in the latency of recurrence and an increase in the metastatic potential of these tumors in the absence of the initiating oncogene. Conversely, forced expression of par-4 significantly delayed tumors recurrence. We next examined the mechanisms by which par-4 inhibits tumor recurrence. Down-regulation of Neu expression led to a dramatic increase in par-4 expression, and suppressing par-4 in primary tumor cells improved their ability to survive Neu down-regulation. Conversely, ectopic expression of par-4 in recurrent tumor cells, which do not express Neu, led to cell death. These results suggest that par-4 loss is required for the ability of cells to survive following oncogene downregulation. Finally, we wished to address whether par-4 regulates tumor relapse in humans. To do this, we analyzed the relationship between par-4 levels and relapse-free survival in women with breast cancer. We found that lower par-4 expression was associated with poor prognosis in several independent patient datasets. Taken together these results identify a cohort of genes, including par-4, that are important regulators of tumor recurrence, and may offer promising targets for therapeutic intervention for the prevention of tumor recurrence.