SUMMARY The tumor stroma is believed to contribute to some of the most malignant characteristics of epithelial tumors. However, signaling between stromal and tumor cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumors. This was associated with the massive remodeling of the extra-cellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumors ameliorated disruption of the tumor microenvironment and was sufficient to decrease tumor growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumor stroma of breast cancer patients. These findings identify the Pten-Ets2 axis as a critical stroma-specific signaling pathway that suppresses mammary epithelial tumors.

The E2F family is conserved from C. elegans to mammals with some family members having transcription activation functions and others having repressor functions 1, 2 . Whereas C. elegans 3 and Drosophila melanogaster 4, 5 have a single E2F activator and repressor proteins, mammals evolved to have at least three activator and five repressor proteins 1,2,6 . Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, we targeted the inactivation of the entire subset of activators, E2f1, E2f2, E2f3a and E2f3b, singly or in combination in mice. We demonstrate that E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus (E2f3a 3bki ; E2f3a 1ki ) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. We conclude that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development. KeywordsE2F3a; E2F3b; Rb; development; proliferation; transcription and apoptosis Since the identification of the founding E2F family member, E2f1 7 , two distinct genes in lower eukaryotes and eight genes in higher eukaryotes have been identified to encode the signature DNA binding domain that endow these transcription factors with E2F 1,2,6 . Among the mammalian E2F activator subset, the E2f3 gene has emerged as the critical family member involved in the control of cell proliferation and development 8,9 . The E2f3 locus was originally thought to encode a single DNA binding activity, but was later shown to drive the expression of two related isoforms, E2f3a and E2f3b, from two distinct promoters 10 . Given the critical link between the E2f3 locus and the control of cell proliferation, we used homologous recombination to individually disrupt its two isoforms in mice and rigorously evaluate how their functions are integrated with that of other E2F activators. The inactivation of E2f3a or E2f3b was achieved by targeting exon 1a or 1b sequences, respectively, using LoxP-cre technology (Fig. 1a). Mice deleted for either exon 1a or exon 1b were identified by Southern blot and genomic PCR analysis (Fig. 1b). Specific ablation of E2f3a or E2f3b was confirmed by Western blot assays using total E2F3-specific antibodies (Fig. 1c).It was previously shown that inactivation of both E2f3a and E2f3b (E2f3 −/− ) in mice with a mixed strain background yielded offspring that developed rather normally 8, 9 , but we show here that breeding these mice into a pure strain background (∼98% pure) resulted in embryonic lethality ( Fig. 1e and Supplementary Fig. 1). Intercrossing E2f3 +/− mice of different pure backgrounds restored viability of E2f3 −/− mice, albeit with some observed strain-specific biase...

Although activation of the STAT3 pathway has been associated with tumor progression in a wide variety of cancer types (including ovarian cancer), the precise mechanism of invasion and metastasis due to STAT3 are not fully delineated in ovarian cancer. We found that pSTAT3 Tyr705 is constitutively activated in patient ascites and ascites-derived ovarian cancer cells (ADOCCs), and the range of STAT3 expression could be very high to low. In vivo transplantation of ADOCCs with high pSTAT3 expression into the ovarian bursa of mice resulted in a large primary tumor and widespread peritoneal metastases as well liver. In contrast, ADOCCs with low STAT3 expression or ADOCCs with STAT3 expression knocked down led to reduced tumor growth and an absence of metastases in vivo. Cytokines derived from the ADOCC culture medium activate the IL-6/STAT pathway in the STAT3 knockout (Ko) cells, compensating for the absence of inherent STAT3 in the cells. Treatment with HO-3867 (a novel STAT3 inhibitor at 100 ppm in an orthotopic murine model) significantly suppressed ovarian tumor growth, angiogenesis, and metastasis by targeting STAT3 and its downstream proteins. HO-3867 was found to have cytotoxic effects in ex-vivo cultures of freshly-collected human ovarian cancers, including those resistant to platinum-based chemotherapy. Our results show that STAT3 is necessary for ovarian tumor progression/metastasis and highlight the potential for targeting STAT3 by HO-3867 as a therapeutic strategy for ovarian cancer.

Germline mutations in the tumor suppressor gene PTEN (phosphatase and tensin homology deleted on chromosome 10) cause Cowden and Bannayan–Riley–Ruvalcaba (BRR) syndromes, two dominantly inherited disorders characterized by mental retardation, multiple hamartomas, and variable cancer risk. Here, we modeled three sentinel mutant alleles of PTEN identified in patients with Cowden syndrome and show that the nonsense Pten ∆4–5 and missense Pten C124R and Pten G129E alleles lacking lipid phosphatase activity cause similar developmental abnormalities but distinct tumor spectra with varying severity and age of onset. Allele-specific differences may be accounted for by loss of function for Pten ∆4–5 , hypomorphic function for Pten C124R , and gain of function for Pten G129E . These data demonstrate that the variable tumor phenotypes observed in patients with Cowden and BRR syndromes can be attributed to specific mutations in PTEN that alter protein function through distinct mechanisms.

STAT3 is well corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (−NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecular modeling studies of this class suggested direct interaction with the STAT3 DNA binding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to be multifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867–mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated from patient ascites, HO-3867 inhibited cell migration/invasion and survival. Our results offer preclinical proof-of-concept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated.