Triple-negative breast cancers (TNBC) are an aggressive disease subtype which unlike other subtypes lack an effective targeted therapy. Inhibitors of the insullin-like growth factor receptor (IGF-1R) have been considered for use in treating TNBC. Here we provide genetic evidence that IGF-1R inhibition promotes development of Wnt1-mediated murine mammary tumors that offer a model of TNBC. We found that in a double transgenic mouse model carrying activated Wnt-1 and mutant IGF-1R, a reduction in IGF-1R signaling reduced tumor latency and promoted more aggressive phenotypes. These tumors displayed a squamal cell phenotype with increased expression of keratins 5/6 and β-catenin. Notably, cell lineage analyses revealed an increase in basal (CD29hi/CD24+) and luminal (CD24+/CD61+/CD29lo) progenitor cell populations, along with increased Nanog expression and decreased Elf5 expression. In these doubly transgenic mice, lung metastases developed with characteristics of the primary tumors, unlike MMTV-Wnt1 mice. Mechanistic investigations showed that pharmacological inhibition of the IGF-1R in vitro was sufficient to increase the tumorsphere-forming efficiency of MMTV-Wnt1 tumor cells. Tumors from doubly transgenic mice also exhibited an increase in the expression ratio of the IGF-II-sensitive, A isoform of the insulin receptor vs the IR-B isoform, which in vitro resulted in enhanced expression of β-catenin. Overall, our results revealed that in Wnt-driven tumors an attenuation of IGF-1R signaling accelerates tumorigenesis and promotes more aggressive phenotypes, with potential implications for understanding TNBC pathobiology and treatment.

Reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency requires the viral transactivator Rta to contact the host protein J recombination signal-binding protein (RBP-J or CSL). RBP-J normally binds DNA sequence-specifically to determine the transcriptional targets of the Notch-signaling pathway, yet Notch alone cannot reactivate KSHV. We previously showed that Rta stimulates RBP-J DNA binding to the viral genome. On a model viral promoter, this function requires Rta to bind to multiple copies of an Rta DNA motif (called "CANT" or Rta-c) proximal to an RBP-J motif. Here, high-resolution ChIP/deep sequencing from infected primary effusion lymphoma cells revealed that RBP-J binds nearly exclusively to different sets of viral genome sites during latency and reactivation. RBP-J bound DNA frequently, but not exclusively, proximal to Rta bound to single, but not multiple, Rta-c motifs. To discover additional regulators of RBP-J DNA binding, we used bioinformatics to identify cellular DNA-binding protein motifs adjacent to either latent or reactivation-specific RBP-J-binding sites. Many of these cellular factors, including POU class homeobox (POU) proteins, have known Notch or herpesvirus phenotypes. Among a set of Rta-and RBP-Jbound promoters, Rta transactivated only those that also contained POU motifs in conserved positions. On some promoters, POU factors appeared to inhibit RBP-J DNA binding unless Rta bound to a proximal Rta-c motif. Moreover, POU2F1/Oct-1 expression was induced during KSHV reactivation, and POU2F1 knockdown diminished infectious virus production. Our results suggest that Rta and POU proteins broadly regulate DNA binding of RBP-J during KSHV reactivation.

The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi’s sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi’s sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. In infected PEL cells, we show that reduction of vDNA synthesis by GSI is associated with gene specific reduction of viral transcription. Specific inhibition of Notch1 by siRNA partially reduces production of infectious KSHV, and ectopic activated Notch (NICD1) enhances virus production induced by Valproic Acid (VPA) treatment. We conclude that constitutive Notch activity is required for robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk complex during viral reactivation.ImportanceKaposi’s sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces expression of specific viral genes yet does not affect growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta’s ability to redistribute RBP-Jk DNA binding to the virus during reactivation.

No abstract

No abstract