The tumor suppressor p53 is lost or mutated in about half of all human cancers and in those tumors where it is wild-type, mechanisms exist to prevent its activation. p53 loss not only prevents incipient tumor cells from undergoing oncogene-induced senescence and apoptosis but also perturbs cell cycle checkpoints. This enables p53-deficient tumor cells with DNA damage to continue cycling, creating a permissive environment for the acquisition of additional mutations. Theoretically, this could contribute to the evolution of a cancer genome that is conducive to metastasis. Importantly, p53 loss also results in the disruption of pathways that inhibit metastasis, and transcriptionally defective TP53 mutants are known to gain additional functions that promote metastasis. Here, we review the evidence supporting a role for p53 loss or mutation in tumor metastasis, with an emphasis on breast cancer.

Estrogen receptor (ER) dimerization is prerequisite for its activation of target gene transcription. Because the two forms of ER, ER␣ and ER␤, exhibit opposing functions in cell proliferation, the ability of ligands to induce ER␣/␤ heterodimers vs. their respective homodimers is expected to have profound impacts on transcriptional outcomes and cellular growth. However, there is a lack of direct methods to monitor the formation of ER␣/␤ heterodimers in vivo and to distinguish the ability of estrogenic ligands to promote ER homo-vs. heterodimerization. Here, we describe bioluminescence resonance energy transfer (BRET) assays for monitoring the formation of ER␣/␤ heterodimers and their respective homodimers in live cells. We demonstrate that although both partners contribute to heterodimerization, ligand-bound ER␣ plays a dominant role. Furthermore, a bioactive component was found to induce ER␤/␤ homodimers, and ER␣/␤ heterodimers but had minimal activity on ER␣/␣ homodimers, posing a model that compounds promoting ER␣/␤ heterodimer formation might have therapeutic value. Thus, ER homodimer and heterodimer BRET assays are applicable to drug screening for dimer-selective selective ER modulators. Furthermore, this strategy can be used to study other nuclear receptor dimers. bioluminescence resonance energy transfer (BRET) ͉ estrogenic ligands ͉ selective estrogen receptor modulator (SERM) ͉ heterodimer ͉ homodimer T he biological actions of estrogens are mediated by estrogen receptors (ERs), which are ligand-inducible transcription factors. Binding of 17␤-estradiol (E 2 ) and other estrogenic compounds triggers receptor dimerization and subsequent association with estrogen response elements (EREs) in the promoter regions of ER-target genes to control gene transcription. ERs exist in two forms, ER␣ and ER␤, which have opposing roles in regulating estrogen action: ER␣ promotes whereas ER␤ inhibits estrogendependent cell growth (1, 2). It has been shown that the coexpression of ER␤ with ER␣ results in reduced ER␣-mediated proliferation of breast cancer cells. Approximately 60% of all breast tumors coexpress ER␣ and ER␤ (3, 4). Despite the findings that the coexpression of ER␤ has been correlated with a more favorable prognosis (5) and decreased biological aggressiveness compared with tumors expressing ER␣ alone (6, 7), whether ER␤ modulates ER␣ by heterodimerization to mediate growth-inhibitory phenotypes remains elusive. Multiple lines of evidence suggest that ER␣/␤ heterodimers do exist in vivo and may function to regulate distinct estrogen-responsive genes (8-10). However, the coexistence of homodimers has prevented a clear understanding of heterodimer function. Hypothetically, different estrogenic ligands could exert different cellular effects via differential induction of ER␣ homodimerization, ER␤ homodimerization, and ER␣/␤ heterodimerization. The differential regulation of these ER subtypes could be influenced by several factors including ligand-binding selectivity, conformational differences upon dimerization, d...

Most triple negative breast cancers (TNBCs) are aggressively metastatic with a high degree of intra-tumoral heterogeneity (ITH), but how ITH contributes to metastasis is unclear. Here, clonal dynamics during metastasis were studied in vivo using two patient-derived xenograft (PDX) models established from the treatment-naive primary breast tumors of TNBC patients diagnosed with synchronous metastasis. Genomic sequencing and high-complexity barcode-mediated clonal tracking reveal robust alterations in clonal architecture between primary tumors and corresponding metastases. Polyclonal seeding and maintenance of heterogeneous populations of low-abundance subclones is observed in each metastasis. However, lung, liver, and brain metastases are enriched for an identical population of high-abundance subclones, demonstrating that primary tumor clones harbor properties enabling them to seed and thrive in multiple organ sites. Further, clones that dominate multi-organ metastases share a genomic lineage. Thus, intrinsic properties of rare primary tumor subclones enable the seeding and colonization of metastases in secondary organs in these models.

Alternative splicing has been shown to causally contribute to the epithelial-mesenchymal transition (EMT) and tumor metastasis. However, the scope of splicing factors that govern alternative splicing in these processes remains largely unexplored. Here we report the identification of A-Kinase Anchor Protein (AKAP8) as a splicing regulatory factor that impedes EMT and breast cancer metastasis. AKAP8 not only is capable of inhibiting splicing activity of the EMT-promoting splicing regulator hnRNPM through protein-protein interaction, it also directly binds to RNA and alters splicing outcomes. Genome-wide analysis shows that AKAP8 promotes an epithelial cell state splicing program. Experimental manipulation of an AKAP8 splicing target CLSTN1 revealed that splice isoform switching of CLSTN1 is crucial for EMT. Moreover, AKAP8 expression and the alternative splicing of CLSTN1 predict breast cancer patient survival. Together, our work demonstrates the essentiality of RNA metabolism that impinges on metastatic breast cancer.

Estrogens play essential roles in the progression of mammary and prostatic diseases. The transcriptional effects of estrogens are transduced by two estrogen receptors, ERα and ERβ, which elicit opposing roles in regulating proliferation: ERα is proliferative while ERβ is anti-proliferative. Exogenous expression of ERβ in ERα-positive cancer cell lines inhibits cell proliferation in response to estrogen and reduces xenografted tumor growth in vivo, suggesting that ERβ might oppose ERα's proliferative effects via formation of ERα/β heterodimers. Despite biochemical and cellular evidence of ERα/β heterodimer formation in cells co-expressing both receptors, the biological roles of the ERα/β heterodimer remain to be elucidated. Here we report the identification of two phytoestrogens that selectively activate ERα/β heterodimers at specific concentrations using a cell-based, two-step high throughput small molecule screen for ER transcriptional activity and ER dimer selectivity. Using ERα/β heterodimer-selective ligands at defined concentrations, we demonstrate that ERα/β heterodimers are growth inhibitory in breast and prostate cells which co-express the two ER isoforms. Furthermore, using Automated Quantitative Analysis (AQUA) to examine nuclear expression of ERα and ERβ in human breast tissue microarrays, we demonstrate that ERα and ERβ are co-expressed in the same cells in breast tumors. The co-expression of ERα and ERβ in the same cells supports the possibility of ERα/β heterodimer formation at physio- and pathological conditions, further suggesting that targeting ERα/β heterodimers might be a novel therapeutic approach to the treatment of cancers which co-express ERα and ERβ.