PTEN PDZ-binding domain suppresses mammary carcinogenesis in the MMTV-PyMT breast cancer model.

Phosphatase and tension homolog (PTEN) is a potent tumor suppressor that possesses a PDZ-binding domain (PDZ-BD) at the end of its carboxyl terminus, whose functions during tumorigenesis remains unclear. Here, we crossed a mouse strain with germline deletion of PTEN PDZ-BD with MMTV-PyMT breast cancer model, and found that knockout (KO) mice display normal development of mammary glands, but have both increased breast tumorigenicity and lung metastasis. Orthotopic allograft experiments suggest the loss of PTEN PDZ-BD in breast cancer cells rather than in tumor microenvironment plays a prominent role in increasing tumor burden. Through RNA-sequencing, we observed a significant downregulation of myoepithelial marker genes in both KO primary breast cancer and orthotopic allografts. Moreover, these myoepithelial marker genes are significantly downregulated in human breast cancer tissues, and are associated with poorer clinical prognosis. In addition, several homeobox genes were also identified to be downreguated in KO breast cancer, whose expressions showed significant positive correlation with myoepithelial marker genes. Overall, our findings suggest a novel tumor suppressive role of PTEN PDZ-BD in a murine model of breast cancer, and the mechanism involves the dysregulation of homeobox genes which may result in defective myoepithelial differentiation in breast cancer cells.

Both canonical and non-canonical Wnt signaling independently promote stem cell growth in mammospheres.

The characterization of mammary stem cells, and signals that regulate their behavior, is of central importance in understanding developmental changes in the mammary gland and possibly for targeting stem-like cells in breast cancer. The canonical Wnt/ß-catenin pathway is a signaling mechanism associated with maintenance of self-renewing stem cells in many tissues, including mammary epithelium, and can be oncogenic when deregulated. Wnt1 and Wnt3a are examples of ligands that activate the canonical pathway. Other Wnt ligands, such as Wnt5a, typically signal via non-canonical, ß-catenin-independent, pathways that in some cases can antagonize canonical signaling. Since the role of non-canonical Wnt signaling in stem cell regulation is not well characterized, we set out to investigate this using mammosphere formation assays that reflect and quantify stem cell properties. Ex vivo mammosphere cultures were established from both wild-type and Wnt1 transgenic mice and were analyzed in response to manipulation of both canonical and non-canonical Wnt signaling. An increased level of mammosphere formation was observed in cultures derived from MMTV-Wnt1 versus wild-type animals, and this was blocked by treatment with Dkk1, a selective inhibitor of canonical Wnt signaling. Consistent with this, we found that a single dose of recombinant Wnt3a was sufficient to increase mammosphere formation in wild-type cultures. Surprisingly, we found that Wnt5a also increased mammosphere formation in these assays. We confirmed that this was not caused by an increase in canonical Wnt/ß-catenin signaling but was instead mediated by non-canonical Wnt signals requiring the receptor tyrosine kinase Ror2 and activity of the Jun N-terminal kinase, JNK. We conclude that both canonical and non-canonical Wnt signals have positive effects promoting stem cell activity in mammosphere assays and that they do so via independent signaling mechanisms.

Mammary stem cells and cancer: roles of Wnt signaling in plain view.

The likely roles of Wnt signaling in regulating mammary stem cell behavior have been much discussed, in part because they may underlie the oncogenic effects of Wnt signaling in mammary tissue. Two recent papers add important data to this field. One tests directly the effects of purified Wnt protein on mouse mammary stem cells in culture and finds a specific increase in the proportion of cells with self-renewing stem cell phenotypes. The second identifies a novel target gene of canonical Wnt signaling that may be expressed in stem cells and is induced in both mouse and human mammary tumors associated with Wnt pathway activation.

Meiotic localization of Mre11 and Rad50 in wild type, spo11-1, and MRN complex mutants of Coprinus cinereus.

The Mre11-Rad50-Nbs1 (MRN) complex is required for numerous cellular processes that involve interactions with DNA double-strand breaks. For the majority of these processes, the MRN complex is thought to act as a unit, with each protein aiding the activity of the others. We have examined the relationship between Mre11 and Rad50 during meiosis in the basidiomycete Coprinus cinereus (Coprinopsis cinerea), investigating to what extent activities of Mre11 and Rad50 are interdependent. We showed that mre11-1 is epistatic to rad50-1 with respect to the time of meiotic arrest, indicating that Mre11 activity facilitates the diffuse diplotene arrest of rad50 mutants. Anti-Mre11 and anti-Rad50 antibodies were used to examine MRN complex localization in a wild-type strain and in spo11, mre11, and rad50 mutants. In wild type, numbers of Mre11 and Rad50 foci peaked at time points corresponding to leptotene and early zygotene. In the spo11-1 mutant, which is defective in meiotic double-strand break formation, foci accumulated throughout prophase I. Of seven MRN mutants examined, only two rad50 strains exhibited Mre11 and Rad50 foci that localized to chromatin, although Mre11 protein was found in the cell for all of them. Analysis of predicted mutant structures showed that stable localization of Mre11 and Rad50 does not depend upon a wild-type hook-proximal coiled coil, but does require the presence of the Rad50 ATPase/adenylate cyclase domains. We found that Mre11 and Rad50 were interdependent for binding to meiotic chromosomes. However, the majority of foci observed apparently contained only one of the two proteins. Independent Mre11 and Rad50 foci might indicate disassociation of the complex during meiosis or could reflect independent structural roles for the two proteins in meiotic chromatin.

Coprinus cinereus rad50 mutants reveal an essential structural role for Rad50 in axial element and synaptonemal complex formation, homolog pairing and meiotic recombination.

The Mre11/Rad50/Nbs1 (MRN) complex is required for eukaryotic DNA double-strand break (DSB) repair and meiotic recombination. We cloned the Coprinus cinereus rad50 gene and showed that it corresponds to the complementation group previously named rad12, identified mutations in 15 rad50 alleles, and mapped two of the mutations onto molecular models of Rad50 structure. We found that C. cinereus rad50 and mre11 mutants arrest in meiosis and that this arrest is Spo11 dependent. In addition, some rad50 alleles form inducible, Spo11-dependent Rad51 foci and therefore must be forming meiotic DSBs. Thus, we think it likely that arrest in both mre11-1 and the collection of rad50 mutants is the result of unrepaired or improperly processed DSBs in the genome and that Rad50 and Mre11 are dispensable in C. cinereus for DSB formation, but required for appropriate DSB processing. We found that the ability of rad50 mutant strains to form Rad51 foci correlates with their ability to promote synaptonemal complex formation and with levels of stable meiotic pairing and that partial pairing, recombination initiation, and synapsis occur in the absence of wild-type Rad50 catalytic domains. Examination of single- and double-mutant strains showed that a spo11 mutation that prevents DSB formation enhances axial element (AE) formation for rad50-4, an allele predicted to encode a protein with intact hook region and hook-proximal coiled coils, but not for rad50-1, an allele predicted to encode a severely truncated protein, or for rad50-5, which encodes a protein whose hook-proximal coiled-coil region is disrupted. Therefore, Rad50 has an essential structural role in the formation of AEs, separate from the DSB-processing activity of the MRN complex.