The Enrichment of Survivin in Exosomes from Breast Cancer Cells Treated with Paclitaxel Promotes Cell Survival and Chemoresistance.

The generation and release of membrane-enclosed packets from cancer cells, called extracellular vesicles (EVs), play important roles in propagating transformed phenotypes, including promoting cell survival. EVs mediate their effects by transferring their contents, which include specific proteins and nucleic acids, to target cells. However, how the cargo and function of EVs change in response to different stimuli remains unclear. Here, we discovered that treating highly aggressive MDAMB231 breast cancer cells with paclitaxel (PTX), a chemotherapy that stabilizes microtubules, causes them to generate a specific class of EV, namely exosomes, that are highly enriched with the cell survival protein and cancer marker, Survivin. Treating MDAMB231 cells with a variety of other chemotherapeutic agents, and inhibitors that block cell growth and survival, did not have the same effect as PTX, with the exception of nocodazole, another inhibitor of microtubule dynamics. Exosomes isolated from PTX-treated MDAMB231 cells strongly promoted the survival of serum-starved and PTX-treated fibroblasts and SKBR3 breast cancer cells, an effect that was ablated when Survivin was knocked-down from these vesicles using siRNA. These findings underscore how the enrichment of a specific cargo in exosomes promotes cell survival, as well as can potentially serve as a marker of PTX resistance.

Microvesicle Cargo and Function Changes upon Induction of Cellular Transformation.

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), have emerged as a major form of intercellular communication, playing important roles in several physiological processes and diseases, including cancer. EVs generated by cancer cells contain a variety of proteins and RNA species that can be transferred between cancer cells as well as between cancer and non-transformed (normal) cells, thereby impacting a number of aspects of cancer progression. Here we show how oncogenic transformation influences the biogenesis and function of EVs using a mouse embryonic fibroblast (MEF) cell line that can be induced to express an oncogenic form of diffuse B cell lymphoma (Dbl). Although MEFs induced to express onco-Dbl generated a similar amount of MVs as uninduced control cells, we found that MVs isolated from onco-Dbl-transformed cells contain a unique signaling protein, the ubiquitously expressed non-receptor tyrosine kinase focal adhesion kinase. The addition of MVs isolated from MEFs expressing onco-Dbl to cultures of fibroblasts strongly promoted their survival and induced their ability to grow under anchorage-independent conditions, outcomes that could be reversed by knocking down focal adhesion kinase and depleting it from the MVs or by inhibiting its kinase activity using a specific inhibitor. We then showed the same to be true for MVs isolated from aggressive MDAMB231 breast cancer cells. Together, these findings demonstrate that the induction of oncogenic transformation gives rise to MVs, which uniquely contain a signaling protein kinase that helps propagate the transformed phenotype and thus may offer a specific diagnostic marker of malignant disease.

Micro RNA responses to chronic or acute exposures to low dose ionizing radiation.

Human health risks of exposure to low dose ionizing radiation remain ambiguous and are the subject of intense debate. A wide variety of biological effects are induced after cellular exposure to ionizing radiation, but the underlying molecular mechanism(s) remain to be completely understood. We hypothesized that low dose γ-radiation-induced effects are controlled by the modulation of micro RNA (miRNA) that participate in the control of gene expression at the posttranscriptional level and are involved in many cellular processes. We monitored the expression of several miRNA in human cells exposed to acute or chronic low doses of 10 cGy or a moderate dose of 400 cGy of (137)Cs γ-rays. Dose, dose rate and time dependent differences in the relative expression of several miRNA were investigated. The expression patterns of many miRNA differed after exposure to either chronic or acute 10 cGy. The expression of miRNA let-7e, a negative regulator of RAS oncogene, and the c-MYC miRNA cluster were upregulated after 10 cGy chronic dose but were downregulated after 3 h of acute 10 cGy. The miR-21 was upregulated in chronic or acute low dose and moderate dose treated cells and its target genes hPDCD4, hPTEN, hSPRY2, and hTPM1 were found to be downregulated. These findings provide evidence that low dose and dose rate γ-irradiation dictate the modulation of miRNA, which can result in a differential cellular response than occurs at high doses. This information will contribute to understanding the risks to human health after exposure to low dose radiation.

α-catenin is a tumor suppressor that controls cell accumulation by regulating the localization and activity of the transcriptional coactivator Yap1.

The Hippo pathway regulates contact inhibition of cell proliferation and, ultimately, organ size in diverse multicellular organisms. Inactivation of the Hippo pathway promotes nuclear localization of the transcriptional coactivator Yap1, a Hippo pathway effector, and can cause cancer. Here, we show that deletion of αE (α epithelial) catenin in the hair follicle stem cell compartment resulted in the development of skin squamous cell carcinoma in mice. Tumor formation was accelerated by simultaneous deletion of αE-catenin and the tumor suppressor-encoding gene p53. A small interfering RNA screen revealed a functional connection between αE-catenin and Yap1. By interacting with Yap1, αE-catenin promoted its cytoplasmic localization, and Yap1 showed constitutive nuclear localization in αE-catenin-null cells. We also found an inverse correlation between αE-catenin abundance and Yap1 activation in human squamous cell carcinoma tumors. These findings identify αE-catenin as a tumor suppressor that inhibits Yap1 activity and sequesters it in the cytoplasm.

Yap1 acts downstream of α-catenin to control epidermal proliferation.

During development and regeneration, proliferation of tissue-specific stem cells is tightly controlled to produce organs of a predetermined size. The molecular determinants of this process remain poorly understood. Here, we investigate the function of Yap1, the transcriptional effector of the Hippo signaling pathway, in skin biology. Using gain- and loss-of-function studies, we show that Yap1 is a critical modulator of epidermal stem cell proliferation and tissue expansion. Yap1 mediates this effect through interaction with TEAD transcription factors. Additionally, our studies reveal that α-catenin, a molecule previously implicated in tumor suppression and cell density sensing in the skin, is an upstream negative regulator of Yap1. α-catenin controls Yap1 activity and phosphorylation by modulating its interaction with 14-3-3 and the PP2A phosphatase. Together, these data identify Yap1 as a determinant of the proliferative capacity of epidermal stem cells and as an important effector of a "crowd control" molecular circuitry in mammalian skin.

Transcriptional modulation of micro-RNA in human cells differing in radiation sensitivity.

PURPOSE: The molecular basis of gene regulation in cells exposed to ionising radiation is not fully understood. Gene regulation occurs at the transcriptional and post-transcriptional levels. Recent studies have suggested that micro-RNA (miRNA) plays a significant role at the post-transcriptional gene regulation. miRNA are a recently identified class of RNA molecules 18-24 nucleotides in length that have been shown to negatively regulate the stability or translation of target messenger RNA. We hypothesised that ionising radiation induced stress response is controlled in part by miRNA and that a difference in tumour protein 53 (p53) status corresponds with altered expression in miRNA responses to ionising radiation. MATERIALS AND METHODS: To test this hypothesis, we investigated the relative expression of several miRNA by quantitative real-time polymerase chain reaction (QPCR) in human cell lines TK6 and WTK1 that differ in p53 status and radiosensitivity after exposure to high and low doses of X-radiation. RESULTS: The suitability of several endogenous miRNA controls was tested for relative quantification by QPCR. The baseline expression of 21 miRNA targets in TK6 and WTK1 cells indicated a wide range of modulation between the two cell lines without exposure to ionising radiation. Differences in the relative expression of miRNA were observed among the two cell lines after radiation treatment. The expression patterns of many miRNA markedly differed within the same cell line after exposure to either 0.5 Gy or 2 Gy doses of X-rays. The expression of eight miRNA belonging to the lethal-7 (let-7) family, which are negative regulators of the rat sarcoma, RAS oncogene, was upregulated in irradiated TK6 cells but was downregulated in WTK1 cells. Alterations in the myelocytomatosis oncogene, c-MYC induced cluster of miRNA were also observed. The micro RNA, miR-15a and miR-16 were upregulated in 0.5 Gy-irradiated TK6 cells but were downregulated after a 2 Gy dose of X-rays. In contrast miR-15 and miR-16 were repressed in 0.5 Gy-exposed WTK1. The miR-21 was upregulated in 0.5 Gy-treated TK6 cells and its target genes programmed cell death factor 4 (hPDCD4) phosphatase and tensin homolog (hPTEN), and sprouty homolog 2 (hSPRY2) were found to be downregulated in these cells. The miR-21 was downregulated in 2 Gy-irradiated TK6 cells, and all three of its target genes were upregulated in 2 Gy-exposed TK6 cells. CONCLUSION: Taken together, these results establish the involvement of miRNA in radiation response and may potentially help explain the mechanisms of gene regulation in the cellular response to ionising radiation exposure.