Breast Tissue Biology Expands the Possibilities for Prevention of Age-Related Breast Cancers.

Preventing breast cancer before it is able to form is an ideal way to stop breast cancer. However, there are limited existing options for prevention of breast cancer. Changes in the breast tissue resulting from the aging process contribute to breast cancer susceptibility and progression and may therefore provide promising targets for prevention. Here, we describe new potential targets, immortalization and inflammaging, that may be useful for prevention of age-related breast cancers. We also summarize existing studies of warfarin and metformin, current drugs used for non-cancerous diseases, that also may be repurposed for breast cancer prevention

TOR Complex 2-Ypk1 Signaling Maintains Sphingolipid Homeostasis by Sensing and Regulating ROS Accumulation

Reactive oxygen species (ROS) are produced during normal metabolism and can function as signaling molecules. However, ROS at elevated levels can damage cells. Here, we identify the conserved target of rapamycin complex 2 (TORC2)/Ypk1 signaling module as an important regulator of ROS in the model eukaryotic organism, S. cerevisiae. We show that TORC2/Ypk1 suppresses ROS produced both by mitochondria as well as by nonmitochondrial sources, including changes in acidification of the vacuole. Furthermore, we link vacuole-related ROS to sphingolipids, essential components of cellular membranes, whose synthesis is also controlled by TORC2/Ypk1 signaling. In total, our data reveal that TORC2/Ypk1 act within a homeostatic feedback loop to maintain sphingolipid levels and that ROS are a critical regulatory signal within this system. Thus, ROS sensing and signaling by TORC2/Ypk1 play a central physiological role in sphingolipid biosynthesis and in the maintenance of cell growth and viability

Abstract 5818: The immortalization process as a therapeutic target

Abstract: Normal human somatic cells have a finite lifespan and intact tumor suppressor barriers, whereas most carcinoma cells have gained immortality and overcome multiple tumor suppressor barriers. We have developed a comprehensive human mammary epithelial cell (HMEC) culture system to examine the alterations involved in the transition from normal finite to malignant immortal; such data can suggest prevention strategies. Our studies have indicated that HMEC initially encounter a stress-associated senescence barrier (stasis) enforced by retinoblastoma (RB). Errors in the RB pathway allow stasis bypass; similar errors are seen early in progression in vivo. Replicative senescence due to telomere erosion is a second extremely stringent barrier, with critically short telomeres leading to genomic instability. Overcoming this barrier requires reactivation of endogenous telomerase, similar to what is seen in high-grade DCIS in vivo. Resulting immortal HMEC are resistant to oncogene-induced-senescence, and exposure to oncogenes that cause finite cells to senesce, can now give rise to malignancy, underscoring the critical importance of the immortalization step in progression. Also like DCIS, the molecular properties of our non/pre-malignant immortal HMEC lines are more similar to malignant immortal than normal finite cells, highlighting the abnormal, cancer-like qualities of immortalized cells. We suggest this similarity is due to a novel process that we have discovered to be involved in cancer-associated immortal transformation, that we have called conversion. HMEC that gain an error permissive for telomerase reactivation still need to undergo additional changes to assume the cancer-associated immortal phenotype: expression of sufficient telomerase activity to maintain short telomeres, mean TRF ~4 kb (which is shorter than what is present in normal finite cells). Conversion appears initiated by a mean TRF of <4 kb, and may entail changes in telomere structure. Possibly, the observed similar alterations in immortal cells ± malignancy results from changes induced by conversion. Importantly, cancer-associated immortalization is unique to cells progressing to cancer, and its inhibition may thus be a valuable therapeutic target. Surprisingly, little has been done to explore this possibility, we believe because: 1) small short-lived animals like mice do not stringently repress telomerase in adult cells; lacking a significant immortalization barrier, they cannot model this critical step in human carcinogenesis; 2) Immortally transformed cells are commonly referred to as normal or untransformed; 3) hTERT immortalization does not model cancer-associated immortalization; there is no conversion to short telomeres. Our preliminary data suggest potential approaches to inhibiting conversion. Citation Format: Martha R. Stampfer, Tara Fresques, Sun-Young Lee, Mark LaBarge, James Garbe. The immortalization process as a therapeutic target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5818

The diversity of nanos expression in echinoderm embryos supports different mechanisms in germ cell specification

Specification of the germ cell lineage is required for sexual reproduction in all animals. However, the timing and mechanisms of germ cell specification is remarkably diverse in animal development. Echinoderms, such as sea urchins and sea stars, are excellent model systems to study the molecular and cellular mechanisms that contribute to germ cell specification. In several echinoderm embryos tested, the germ cell factor Vasa accumulates broadly during early development and is restricted after gastrulation to cells that contribute to the germ cell lineage. In the sea urchin, however, the germ cell factor Vasa is restricted to a specific lineage by the 32-cell stage. We therefore hypothesized that the germ cell specification program in the sea urchin/Euechinoid lineage has evolved to an earlier developmental time point. To test this hypothesis we determined the expression pattern of a second germ cell factor, Nanos, in four out of five extant echinoderm clades. Here we find that Nanos mRNA does not accumulate until the blastula stage or later during the development of all other echinoderm embryos except those that belong to the Echinoid lineage. Instead, Nanos is expressed in a restricted domain at the 32-128 cell stage in Echinoid embryos. Our results support the model that the germ cell specification program underwent a heterochronic shift in the Echinoid lineage. A comparison of Echinoid and non-Echinoid germ cell specification mechanisms will contribute to our understanding of how these mechanisms have changed during animal evolution

The diversity of nanos expression in echinoderm embryos supports different mechanisms in germ cell specification

Specification of the germ cell lineage is required for sexual reproduction in all animals. However, the timing and mechanisms of germ cell specification is remarkably diverse in animal development. Echinoderms, such as sea urchins and sea stars, are excellent model systems to study the molecular and cellular mechanisms that contribute to germ cell specification. In several echinoderm embryos tested, the germ cell factor Vasa accumulates broadly during early development and is restricted after gastrulation to cells that contribute to the germ cell lineage. In the sea urchin, however, the germ cell factor Vasa is restricted to a specific lineage by the 32-cell stage. We therefore hypothesized that the germ cell specification program in the sea urchin/Euechinoid lineage has evolved to an earlier developmental time point. To test this hypothesis we determined the expression pattern of a second germ cell factor, Nanos, in four out of five extant echinoderm clades. Here we find that Nanos mRNA does not accumulate until the blastula stage or later during the development of all other echinoderm embryos except those that belong to the Echinoid lineage. Instead, Nanos is expressed in a restricted domain at the 32–128 cell stage in Echinoid embryos. Our results support the model that the germ cell specification program underwent a heterochronic shift in the Echinoid lineage. A comparison of Echinoid and non-Echinoid germ cell specification mechanisms will contribute to our understanding of how these mechanisms have changed during animal evolution

Selective ATP-Competitive Inhibitors of TOR Suppress Rapamycin-Insensitive Function of TORC2 in <i>Saccharomyces cerevisiae</i>

The target of rapamycin (TOR) is a critical regulator of growth, survival, and energy metabolism. The allosteric TORC1 inhibitor rapamycin has been used extensively to elucidate the TOR related signal pathway but is limited by its inability to inhibit TORC2. We used an unbiased cell proliferation assay of a kinase inhibitor library to discover QL-IX-55 as a potent inhibitor of S. <i>cerevisiae</i> growth. The functional target of QL-IX-55 is the ATP-binding site of TOR2 as evidenced by the discovery of resistant alleles of TOR2 through rational design and unbiased selection strategies. QL-IX-55 is capable of potently inhibiting both TOR complex 1 and 2 (TORC1 and TORC2) as demonstrated by biochemical IP kinase assays (IC₅₀ <50 nM) and cellular assays for inhibition of substrate YPK1 phosphorylation. In contrast to rapamycin, QL-IX-55 is capable of inhibiting TORC2-dependent transcription, which suggests that this compound will be a powerful probe to dissect the Tor2/TORC2-related signaling pathway in yeast

Selective ATP-Competitive Inhibitors of TOR Suppress Rapamycin-Insensitive Function of TORC2 in <i>Saccharomyces cerevisiae</i>

The target of rapamycin (TOR) is a critical regulator of growth, survival, and energy metabolism. The allosteric TORC1 inhibitor rapamycin has been used extensively to elucidate the TOR related signal pathway but is limited by its inability to inhibit TORC2. We used an unbiased cell proliferation assay of a kinase inhibitor library to discover QL-IX-55 as a potent inhibitor of S. <i>cerevisiae</i> growth. The functional target of QL-IX-55 is the ATP-binding site of TOR2 as evidenced by the discovery of resistant alleles of TOR2 through rational design and unbiased selection strategies. QL-IX-55 is capable of potently inhibiting both TOR complex 1 and 2 (TORC1 and TORC2) as demonstrated by biochemical IP kinase assays (IC₅₀ <50 nM) and cellular assays for inhibition of substrate YPK1 phosphorylation. In contrast to rapamycin, QL-IX-55 is capable of inhibiting TORC2-dependent transcription, which suggests that this compound will be a powerful probe to dissect the Tor2/TORC2-related signaling pathway in yeast