Omental preadipocytes stimulate matrix remodeling and IGF signaling to support ovarian cancer metastasis.

Ovarian cancer can metastasize to the omentum, which is associated with a complex tumor microenvironment. Omental stromal cells facilitate ovarian cancer colonization by secreting cytokines and growth factors. Improved understanding of the tumor supportive functions of specific cell populations in the omentum could identify strategies to prevent and treat ovarian cancer metastasis. Here, we showed that omental preadipocytes enhance the tumor initiation capacity of ovarian cancer cells. Secreted factors from preadipocytes supported cancer cell viability during nutrient and isolation stress and enabled prolonged proliferation. Co-culturing with pre-adipocytes led to upregulation of genes involved in extracellular matrix (ECM) organization, cellular response to stress, and regulation of insulin-like growth factor (IGF) signaling in ovarian cancer cells. IGF-1 induced ECM genes and increased alternative NF-κB signaling by activating RelB. Inhibiting the IGF-1 receptor (IGF1R) initially increased tumor omental adhesion but decreased growth of established preadipocyte-induced subcutaneous tumors as well as established intraperitoneal tumors. Together, this study shows that omental preadipocytes support ovarian cancer progression, which has implications for targeting metastasis.

Phosphorylation of the Myogenic Factor Myocyte Enhancer Factor-2 Impacts Myogenesis In Vivo.

Activity of the myogenic regulatory protein myocyte enhancer factor-2 (MEF2) is modulated by post-translational modification. We investigated the in vivo phosphorylation of Drosophila MEF2, and identified serine 98 (S98) as a phosphorylated residue. Phospho-mimetic (S98E) and phospho-null (S98A) isoforms of MEF2 did not differ from wild-type in their activity in vitro, so we used CRISPR/Cas9 to generate an S98A allele of the endogenous gene. In mutant larvae we observed phenotypes characteristic of reduced MEF2 function, including reduced body wall muscle size and reduced expression of myofibrillar protein genes; conversely,S98A homozygotes showed enhanced MEF2 function through muscle differentiation within the adult myoblasts associated with the wing imaginal disc. In adults, S98A homozygotes were viable with normal mobility, yet showed patterning defects in muscles that were enhanced when the S98A allele was combined with a Mef2 null allele. Overall our data indicate that blocking MEF2 S98 phosphorylation in myoblasts enhances its myogenic capability, whereas blocking S98 phosphorylation in differentiating muscles attenuates MEF2 function. Our studies are among the first to assess the functional significance of MEF2 phosphorylation sites in the intact animal, and suggest that the same modification can have profoundly different effects upon MEF2 function depending upon the developmental context.

TWEAK-Fn14-RelB Signaling Cascade Promotes Stem Cell-like Features that Contribute to Post-Chemotherapy Ovarian Cancer Relapse.

Disease recurrence in high-grade serous ovarian cancer may be due to cancer stem-like cells (CSC) that are resistant to chemotherapy and capable of reestablishing heterogeneous tumors. The alternative NF-κB signaling pathway is implicated in this process; however, the mechanism is unknown. Here we show that TNF-like weak inducer of apoptosis (TWEAK) and its receptor, Fn14, are strong inducers of alternative NF-κB signaling and are enriched in ovarian tumors following chemotherapy treatment. We further show that TWEAK enhances spheroid formation ability, asymmetric division capacity, and expression of SOX2 and epithelial-to-mesenchymal transition genes VIM and ZEB1 in ovarian cancer cells, phenotypes that are enhanced when TWEAK is combined with carboplatin. Moreover, TWEAK in combination with chemotherapy induces expression of the CSC marker CD117 in CD117- cells. Blocking the TWEAK-Fn14-RelB signaling cascade with a small-molecule inhibitor of Fn14 prolongs survival following carboplatin chemotherapy in a mouse model of ovarian cancer. These data provide new insights into ovarian cancer CSC biology and highlight a signaling axis that should be explored for therapeutic development. IMPLICATIONS: This study identifies a unique mechanism for the induction of ovarian cancer stem cells that may serve as a novel therapeutic target for preventing relapse.

Insulin-like growth factor binding protein 5: Diverse roles in cancer.

Insulin-like growth factor binding proteins (IGFBPs) and the associated signaling components in the insulin-like growth factor (IGF) pathway regulate cell differentiation, proliferation, apoptosis, and adhesion. Of the IGFBPs, insulin-like growth factor binding protein 5 (IGFBP5) is the most evolutionarily conserved with a dynamic range of IGF-dependent and -independent functions, and studies on the actions of IGFBP5 in cancer have been somewhat paradoxical. In cancer, the IGFBPs respond to external stimuli to modulate disease progression and therapeutic responsiveness in a context specific manner. This review discusses the different roles of IGF signaling and IGFBP5 in disease with an emphasis on discoveries within the last twenty years, which underscore a need to clarify the IGF-independent actions of IGFBP5, the impact of its subcellular localization, the differential activities of each of the subdomains, and the response to elements of the tumor microenvironment (TME). Additionally, recent advances addressing the role of IGFBP5 in resistance to cancer therapeutics will be discussed. A better understanding of the contexts in which IGFBP5 functions will facilitate the discovery of new mechanisms of cancer progression that may lead to novel therapeutic opportunities.

Characterization of SOX2, OCT4 and NANOG in Ovarian Cancer Tumor-Initiating Cells.

The identification of tumor-initiating cells (TICs) has traditionally relied on surface markers including CD133, CD44, CD117, and the aldehyde dehydrogenase (ALDH) enzyme, which have diverse expression across samples. A more reliable indication of TICs may include the expression of embryonic transcription factors that support long-term self-renewal, multipotency, and quiescence. We hypothesize that SOX2, OCT4, and NANOG will be enriched in ovarian TICs and may indicate TICs with high relapse potential. We evaluated a panel of eight ovarian cancer cell lines grown in standard 2-D culture or in spheroid-enriching 3-D culture, and correlated expression with growth characteristics, TIC marker expression, and chemotherapy resistance. RNA-sequencing showed that cell cycle regulation pathways involving SOX2 were elevated in 3-D conditions. HGSOC lines had longer doubling-times, greater chemoresistance, and significantly increased expression of SOX2, OCT4, and NANOG in 3-D conditions. CD117+ or ALDH+/CD133+ cells had increased SOX2, OCT4, and NANOG expression. Limiting dilution in in vivo experiments implicated SOX2, but not OCT4 or NANOG, with early tumor-initiation. An analysis of patient data suggested a stronger role for SOX2, relative to OCT4 or NANOG, for tumor relapse potential. Overall, our findings suggest that SOX2 may be a more consistent indicator of ovarian TICs that contribute to tumor repopulation following chemotherapy. Future studies evaluating SOX2 in TIC biology will increase our understanding of the mechanisms that drive ovarian cancer relapse.

Stat2 loss leads to cytokine-independent, cell-mediated lethality in LPS-induced sepsis.

Deregulated Toll-like receptor (TLR)-triggered inflammatory responses that depend on NF-κB are detrimental to the host via excessive production of proinflammatory cytokines, including TNF-α. Stat2 is a critical component of type I IFN signaling, but it is not thought to participate in TLR signaling. Our study shows that LPS-induced lethality in Stat2(-/-) mice is accelerated as a result of increased cellular transmigration. Blocking intercellular adhesion molecule-1 prevents cellular egress and confers survival of Stat2(-/-) mice. The main determinant of cellular egress in Stat2(-/-) mice is the genotype of the host and not the circulating leukocyte. Surprisingly, lethality and cellular egress observed on Stat2(-/-) mice are not associated with excessive increases in classical sepsis cytokines or chemokines. Indeed, in the absence of Stat2, cytokine production in response to multiple TLR agonists is reduced. We find that Stat2 loss leads to reduced expression of NF-κB target genes by affecting nuclear translocation of NF-κB. Thus, our data reveal the existence of a different mechanism of LPS-induced lethality that is independent of NF-κB triggered cytokine storm but dependent on cellular egress.

Characterization of complex B cell epitopes on woodchuck hepatitis virus surface antigens by using plasmids encoding chimeric proteins and DNA immunization.

The conformational nature of the B cell epitopes on the hepadnavirus surface antigens makes its characterization difficult. Here, a new approach by DNA vaccination with plasmids expressing chimeric hepadnavirus surface antigens was explored to determine B cell epitopes on the surface antigens of woodchuck hepatitis virus (WHsAg). A series of chimeric genes consisting of complementary fragments of WHsAg and hepatitis B virus surface antigens (HBsAg) was constructed. These plasmids expressed the following: (i) middle chimeric surface antigens (MCSAgs), including pre-S2 region and small surface antigens; (ii) small chimeric surface antigens (CSAgs); (iii) a mutated WHsAg with two amino acid substitutions, the Leu 136 to Thr and Ala 140 to Asp, within the central immunogenic region. The mutated region from amino acid 135 to 143 within WHsAg mimics the second loop of the HBsAg a-determinant. MCSAgs and CSAgs were expressed in transiently transfected mammalian cells and were reactive to anti-HBsAg and anti-WHsAg, as shown by indirect immunofluorescence staining and ELISA. Vaccination with plasmids encoding MCSAgs induced strong antibody responses to the pre-S2 region. Anti-pre-S2 antibodies were directed to a linear, immunodominant region within the amino-terminal region of the pre-S2 region and were able to precipitate serum WHsAg. Vaccinations with the plasmids expressing the CSAgs led to the conclusion that an extended region aa 116-169 of WHsAg, analogous to the HBsAg a-determinant, was sufficient for the induction of anti-WHsAg antibodies. The mutated WHsAg with the second loop of the HBsAg a-determinant efficiently induced anti-WHsAg antibodies, but also a low titer of anti-HBsAg. Thus, multiple B cell epitopes of a linear and conformational nature are present on WHsAg. We presented an efficient and broadly applicable strategy for analysis of complex immunogenic determinants of natural or mutated viral antigens.