Perivascular cell-specific knockout of the stem cell pluripotency gene Oct4 inhibits angiogenesis.

The stem cell pluripotency factor Oct4 serves a critical protective role during atherosclerotic plaque development by promoting smooth muscle cell (SMC) investment. Here, we show using Myh11-CreERT2 lineage-tracing with inducible SMC and pericyte (SMC-P) knockout of Oct4 that Oct4 regulates perivascular cell migration and recruitment during angiogenesis. Knockout of Oct4 in perivascular cells significantly impairs perivascular cell migration, increases perivascular cell death, delays endothelial cell migration, and promotes vascular leakage following corneal angiogenic stimulus. Knockout of Oct4 in perivascular cells also impairs perfusion recovery and decreases angiogenesis following hindlimb ischemia. Transcriptomic analyses demonstrate that expression of the migratory gene Slit3 is reduced following loss of Oct4 in cultured SMCs, and in Oct4-deficient perivascular cells in ischemic hindlimb muscle. Together, these results provide evidence that Oct4 plays an essential role within perivascular cells in injury- and hypoxia-induced angiogenesis.

Knockdown of Long Noncoding RNA POU5F1B Promotes Radiosensitivity in Esophageal Carcinoma.

BACKGROUND POU5F1B, serving as a carcinogen, participates in radiosensitivity of several tumors. However, in esophageal cancer, its potential mechanism and function in regulating radiosensitivity remain unclear. MATERIAL AND METHODS The expression level of POU5F1B was detected in plasma of esophageal tumor patients and cancer cell lines. The effect of POU5F1B knockdown on cell proliferation and colony formation was determined using CCK-8 assay and colony formation assay. Cell apoptosis rate was detected by flow cytometry. RESULTS POU5F1B expression level declined after radiotherapy in the plasma of esophageal cancer patients (p=0.025). Compared with HEEPIC, the level of POU5F1B was upregulated in ECA109 (p<0.01), ECA9706 (p<0.01), KYSE410 (p<0.01), and KYSE510 (p=0.036). The silencing of POU5F1B played a role in inhibiting colony formation. After radiotherapy, the apoptosis rates in the ECA109 with 4Gy si-POU5F1B group and 4Gy si-NC group were 39.1±0.1% and 35.3±0.1%, respectively (p=0.0193). The rate was 21.00±0.1 and 29.1±0.1% (p<0.0072) in the si-NC group and si-POU5F1B group, respectively. For proliferation rate, 4Gy si-POU5F1B ECA109 performed better than 4Gy si-NC. CONCLUSIONS Radiotherapy contributed to the decline in the expression level of POU5F1B in plasma, which was upregulated in ECA109, ECA9706, KYSE410, and KYSE510, but not in HEEPIC. The knockdown of POU5F1B increased the radiosensitivity of esophageal cancer cell lines.

Knockdown of stem cell regulator Oct4A in ovarian cancer reveals cellular reprogramming associated with key regulators of cytoskeleton-extracellular matrix remodelling.

Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas.

Genomic evolution and chemoresistance in germ-cell tumours.

Germ-cell tumours (GCTs) are derived from germ cells and occur most frequently in the testes. GCTs are histologically heterogeneous and distinctly curable with chemotherapy. Gains of chromosome arm 12p and aneuploidy are nearly universal in GCTs, but specific somatic genomic features driving tumour initiation, chemosensitivity and progression are incompletely characterized. Here, using clinical whole-exome and transcriptome sequencing of precursor, primary (testicular and mediastinal) and chemoresistant metastatic human GCTs, we show that the primary somatic feature of GCTs is highly recurrent chromosome arm level amplifications and reciprocal deletions (reciprocal loss of heterozygosity), variations that are significantly enriched in GCTs compared to 19 other cancer types. These tumours also acquire KRAS mutations during the development from precursor to primary disease, and primary testicular GCTs (TGCTs) are uniformly wild type for TP53. In addition, by functional measurement of apoptotic signalling (BH3 profiling) of fresh tumour and adjacent tissue, we find that primary TGCTs have high mitochondrial priming that facilitates chemotherapy-induced apoptosis. Finally, by phylogenetic analysis of serial TGCTs that emerge with chemotherapy resistance, we show how TGCTs gain additional reciprocal loss of heterozygosity and that this is associated with loss of pluripotency markers (NANOG and POU5F1) in chemoresistant teratomas or transformed carcinomas. Our results demonstrate the distinct genomic features underlying the origins of this disease and associated with the chemosensitivity phenotype, as well as the rare progression to chemoresistance. These results identify the convergence of cancer genomics, mitochondrial priming and GCT evolution, and may provide insights into chemosensitivity and resistance in other cancers.

Ionizing radiation induces stemness in cancer cells.

The cancer stem cell (CSC) model posits the presence of a small number of CSCs in the heterogeneous cancer cell population that are ultimately responsible for tumor initiation, as well as cancer recurrence and metastasis. CSCs have been isolated from a variety of human cancers and are able to generate a hierarchical and heterogeneous cancer cell population. CSCs are also resistant to conventional chemo- and radio-therapies. Here we report that ionizing radiation can induce stem cell-like properties in heterogeneous cancer cells. Exposure of non-stem cancer cells to ionizing radiation enhanced spherogenesis, and this was accompanied by upregulation of the pluripotency genes Sox2 and Oct3/4. Knockdown of Sox2 or Oct3/4 inhibited radiation-induced spherogenesis and increased cellular sensitivity to radiation. These data demonstrate that ionizing radiation can activate stemness pathways in heterogeneous cancer cells, resulting in the enrichment of a CSC subpopulation with higher resistance to radiotherapy.

Histological evidence for the existence of germ cell tumor cells showing embryonal carcinoma morphology but lacking OCT4 expression and cisplatin sensitivity.

The biological basis for manifestation of chemotherapy resistance in metastatic testicular germ cell tumors (GCT) remains obscure and is of particular clinical interest. In nonseminomatous GCT (NSGCT) the pluripotent embryonal carcinoma (EC) cells are the precursors of the manifold differentiated structures but also drive the malignant growth. They are known to be hypersensitive towards DNA-damaging agents and to express the embryonal transcription factor OCT4. We recently characterized EC cells that lack OCT4 expression and show cisplatin resistance. In the present, immunohistochemical study we analyzed the composition of NSGCT with the focus on such OCT4-negative EC cells using a NSGCT xenograft model as well as patient-derived NSGCT samples. In the xenograft model, the cisplatin-sensitive cell line H12.1 gives rise to xenografts where EC structures are mainly composed of OCT4-positive cells, whereas xenografts from the resistant cell line 1411HP exclusively comprise OCT4-negative EC areas. We found that post-chemotherapy residual metastatic tumors of patients can be comprised of exclusively OCT4-negative EC, whereas the matched testicular primary tumor harbors OCT4-positive EC. Thorough histological analyses revealed a few examples of such OCT4-negative EC cells also in the testicular primary tumor as well as in xenografts from the cisplatin-sensitive NSGCT-cell line. For these cells we propose an identity as early extraembryonal progenitor cells directly derived from OCT4-expressing EC cells. This challenges the use of the term EC cell. The data also support our hypothesis that malignant growth of resistant NSGCT may be driven by this cell type.

Expression of Scl in mesoderm rescues hematopoiesis in the absence of Oct-4.

In embryonic stem cells, Oct-4 concentration is critical in determining the development of endoderm, mesoderm, and trophectoderm. Although Oct-4 expression is essential for mesoderm development, it is unclear whether it has a role in the development of specific mesodermal tissues. In this study, we have examined the importance of Oct-4 in the generation of hematopoietic cells using an inducible Oct-4 ESC line. We demonstrate that Oct-4 has a role in supporting hematopoiesis after specifying brachyury-positive mesoderm. When we suppressed Oct-4 expression before or after mesoderm specification, no hematopoietic cells are detected. However, hematopoiesis can be rescued in the absence of Oct-4 after mesoderm specification if the essential hematopoietic transcription factor stem cell leukemia is expressed. Our results suggest that, for hematopoiesis to occur, Oct-4 is required for the initial specification of mesoderm and subsequently is required for the development of hematopoietic cells from uncommitted mesoderm.

Alterations of the cytoskeleton in all three embryonic lineages contribute to the epiboly defect of Pou5f1/Oct4 deficient MZspg zebrafish embryos.

Pou5f1/Oct4 is a transcription factor required for pluripotency of embryonic stem cells in mammals. Zebrafish pou5f1 deficient maternal and zygotic spiel ohne grenzen (MZspg) mutant embryos develop severe gastrulation defects, are dorsalized, and defective in endoderm formation. Here we analyze in detail gastrulation defects, which are manifested by a severe delay in epiboly progression. All three embryonic lineages in MZspg embryos behave abnormally during epiboly: the yolk cell forms an altered array of cortical microtubules and F-Actin, with large patches of microtubule free areas; the enveloping layer (EVL) is delayed in the coordinated cell shape changes of marginal cells, that may be mediated by F-Actin; the deep layer cells (DEL), forming the embryo proper, are non-autonomously affected in their motility and do not enter the space opening by epiboly of the EVL. Analysis of adhesiveness as well as high resolution in vivo time lapse image analysis of DEL cells suggests changed adhesive properties and inability to migrate properly on EVL and yolk syncytial layer (YSL) surfaces. Our data further reveal that during epiboly the EVL may actively probe the YSL by filopodia formation, rather than just being passively pulled vegetalwards. Our findings on the effect of Pou5f1 on cell behavior may be relevant to understand stem cell behavior and tumorigenesis involving Pou5f1.

LIF-mediated control of embryonic stem cell self-renewal emerges due to an autoregulatory loop.

Stem cells convert graded stimuli into all-or-nothing cell-fate responses. We investigated how embryonic stem cells (ESCs) convert leukemia inhibitory factor (LIF) concentration into an all-or-nothing cell-fate decision (self-renewal). Using a combined experimental/computational approach we demonstrate unexpected switch-like (on/off) signaling in response to LIF. This behavior emerges over time due to a positive feedback loop controlling transcriptional expression of LIF signaling pathway components. The autoregulatory loop maintains robust pathway responsiveness ("on") at sufficient concentrations of exogenous LIF, while autocrine signaling and low concentrations of exogenous LIF cause ESCs to adopt the weakly responsive ("off") state of differentiated cells. We demonstrate that loss of ligand responsiveness is reversible and precedes loss of the ESC transcription factors Oct4 and Nanog, suggesting an early step in the hierarchical control of differentiation. While endogenously produced ligands were insufficient to sustain the "on" state, they buffer it, influencing the timing of differentiation. These results demonstrate a novel switch-like behavior, which establishes the LIF threshold for ESC self-renewal.