Expression of R-Spondin 1 in Apc(Min/+) Mice Suppresses Growth of Intestinal Adenomas by Altering Wnt and Transforming Growth Factor Beta Signaling

BACKGROUND & AIMS: Mutations in the APC gene and other genes in the Wnt signaling pathway contribute to development of colorectal carcinomas. R-spondins (RSPOs) are secreted proteins that amplify Wnt signaling in intestinal stem cells. Alterations in RSPO genes have been identified in human colorectal tumors. We studied the effects of RSPO1 overexpression in ApcMin/thorn mutant mice. METHODS: An adeno associated viral vector encoding RSPO1-Fc fusion protein, or control vector, was injected into ApcMin/thornmice. Their intestinal crypts were isolated and cultured as organoids. which were incubated with or without RSPO1-Fc and an inhibitor of transforming growth factor beta receptor (TGFBR). Livers were collected from mice and analyzed by immunohistochemistry. Organoids and adenomas were analyzed by quantitative reverse-transcription PCR, single cell RNA sequencing, and immunohistochemistry. RESULTS: Intestines from Apcthorn/thorn mice injected with the vector encoding RSPO1-Fc had significantly deeper crypts, longer villi, with increased EdU labeling, indicating increased proliferation of epithelial cells, in comparison to mice given control vector. AAV-RSPO1-Fctransduced ApcMin/thorn mice also developed fewer and smaller intestinal tumors and had significantly longer survival times. Adenomas of ApcMin/thorn mice injected with the RSPO1-Fc vector showed a rapid increase in apoptosis and in the expression of Wnt target genes, followed by reduced expression of messenger RNAs and proteins regulated by the Wnt pathway, reduced cell proliferation, and less crypt branching than adenomas of mice given the control vector. Addition of RSPO1 reduced the number of adenoma organoids derived from ApcMin/thorn mice and suppressed expression of Wnt target genes but increased phosphorylation of SMAD2 and transcription of genes regulated by SMAD. Inhibition of TGFBR signaling in organoids stimulated with RSPO1-Fc restored organoid formation and expression of genes regulated by Wnt. The TGFBR inhibitor restored apoptosis in adenomas from ApcMin/thorn mice expressing RSPO1Fc back to the same level as in the adenomas from mice given the control vector. CONCLUSIONS: Expression of RSPO1 in ApcMin/thorn mice increases apoptosis and reduces proliferation and Wnt signaling in adenoma cells, resulting in development of fewer and smaller intestinal tumors and longer mouse survival. Addition of RSPO1 to organoids derived from adenomas inhibits their growth and promotes proliferation of intestinal stem cells that retain the APC protein; these effects are reversed by TGFB inhibitor. Strategies to increase the expression of RSPO1 might be developed for the treatment of intestinal adenomas.Peer reviewe

Serotype-Related Variation in Susceptibility to Complement Deposition and Opsonophagocytosis among Clinical Isolates of Streptococcus pneumoniae ▿

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae; it affects complement resistance and shields the bacterium from phagocytes. Certain capsular serotypes appear to be better able to cause invasive disease than others. Serotypes 1 and 5 are common causes of invasive disease but are rarely isolated from healthy carriers, whereas serotypes 6B and 23F are more frequently isolated from carriage than invasive disease. We have recently shown that serotypes 6B and 19F differ in resistance to complement C3 deposition and opsonophagocytic killing. In this study we assessed the complement resistance and susceptibility to opsonophagocytosis of several other serotypes targeted by the pneumococcal conjugate vaccines. Clinical isolates of serotypes 1, 4, 5, 14, 18C, and 23F were tested along reference strains of corresponding capsular types. The concentration of anticapsular antibodies required for opsonophagocytic killing correlated inversely with C3 deposition on the serotype. Serotype 1 was the most resistant of the clinical isolates to C3 deposition and, along with serotypes 5 and 19F, required the highest concentration of capsule antibodies for opsonophagocytic killing, whereas serotype 23F was the most sensitive to opsonophagocytosis. Sensitivity to C3 deposition and opsonophagocytosis was associated with serotype-specific mortality of invasive pneumococcal disease, suggesting that the primary pathogens, such as serotypes 1 and 5, are more resistant to complement and require a higher concentration of capsule antibodies for opsonophagocytic killing than the opportunistic serotypes such as 6B and 23F, which are associated with a more severe disease outcome

uPARAP/Endo180 receptor is a gatekeeper of VEGFR-2/VEGFR-3 heterodimerisation during pathological lymphangiogenesis

The development of new lymphatic vessels occurs in many cancerous and inflammatory diseases through the binding of VEGF-C to its receptors, VEGFR-2 and VEGFR-3. The regulation of VEGFR-2/VEGFR-3 heterodimerisation and its downstream signaling in lymphatic endothelial cells (LECs) remain poorly understood. Here, we identify the endocytic receptor, uPARAP, as a partner of VEGFR-2 and VEGFR-3 that regulates their heterodimerisation. Genetic ablation of uPARAP leads to hyperbranched lymphatic vasculatures in pathological conditions without affecting concomitant angiogenesis. In vitro, uPARAP controls LEC migration in response to VEGF-C but not VEGF-A or VEGF-CCys156Ser. uPARAP restricts VEGFR-2/VEGFR-3 heterodimerisation and subsequent VEGFR-2-mediated phosphorylation and inactivation of Crk-II adaptor. uPARAP promotes VEGFR-3 signaling through the Crk-II/JNK/paxillin/Rac1 pathway. Pharmacological Rac1 inhibition in uPARAP knockout mice restores the wild-type phenotype. In summary, our study identifies a molecular regulator of lymphangiogenesis, and uncovers novel molecular features of VEGFR-2/VEGFR-3 crosstalk and downstream signaling during VEGF-C-driven LEC sprouting in pathological conditions.Peer reviewe

Expression of R-spondin1 in ApcMin/+ Mice Reduces Growth of Intestinal Adenomas by Altering Wnt and TGFB Signaling

Suolistosyövät ovat läntisen maailman kolmanneksi yleisimpiä syöpiä. Alati kehittyvistä hoitomuodoista huolimatta edelleenkin joka kolmas suolistosyövistä leviää muihin elimiin, ja merkittävä osa potilaista menehtyy. Yleisesti tiedetään, että syöpäsolut vaativat jakautuakseen useita erilaisia mutaatioita, joiden ansiosta ne pääsevät karkaamaan elimistön kontrollista. Suolistosyövissä tyypillinen mutaatio on solunsisäistä Wnt-signalointireittiä säätelevässä APC-geenissä. APC:n periytyvä mutaatio aiheuttaa Familial Adenomatous Polyposis (FAP)-nimisen sairauden, jossa potilaan suolistoon kehittyy jopa satoja adenoomia. Nämä etenevät hoitamattomana syöväksi lähes 100% todennäköisyydellä. Tämän tutkielman tarkoituksena oli selvittää tarkemmin suolistosyövän kantasolujen ja niitä säätelevien, Wnt-signalointia vahvistavien kasvutekijöiden, R-spondiinien (RSPO), toimintaa, erityisesti kasvainten kehityksen alkuvaiheissa. Tätä tarkoitusta varten tuotettiin AAV-vektori, jonka avulla voitiin käynnistää systeeminen R-spondiinituotanto suolistosyövän Apc-mutantissa hiirimallissa. R-spondiinien tiedetään edistävän suoliston terveiden kantasolujen toimintaa, ja niiden on ajateltu olevan merkittävässä roolissa myös suolistosyöpien kehityksessä. AAV-RSPO1-vektori aikaansaikin villityypin hiirissä odotetusti suolen kantasolujen toiminnan kiihtymisen. Kuitenkin hiiren suolistokasvaimissa vaikutus oli päinvastainen; RSPO1-käsittely hidasti ApcMin-hiirten adenoomien kasvua, kiihdytti apoptoosia spesifisti adenoomasoluissa sekä pidensi kasvaimista kärsivien hiirten elinikää verrokkeihin nähden merkittävästi. Mekanistisissa tutkimuksissa kävi ilmi, että AAV-RSPO1-käsittely aktivoi TGFB-signalointireitin, jonka tiedetään aktivoivan solunsisäistä apoptoosikoneistoa yksinomaan kasvainsoluissa, ja siten rajoittavan niiden kasvua. Tämän seurauksena suolen terveet solut saavuttivat kilpailuedun kasvainsoluihin nähden, ja syrjäyttivät kasvainsoluja, jolloin adenoomista päästiin eroon lähes kokonaan. Näiden löydösten perusteella voisikin olla mahdollista kehittää uusia hoitomuotoja suolistosyöpiin, erityisesti FAP:tä sairastaville potilaille

Oncogenic herpesvirus engages endothelial transcription factors SOX18 and PROX1 to increase viral genome copies and virus production

Kaposi sarcoma is a tumor caused by Kaposi sarcoma herpesvirus (KSHV) infection and is thought to originate from lymphatic endothelial cells (LEC). While KSHV establishes latency in virtually all susceptible cell types, LECs support spontaneous expression of oncogenic lytic genes, high viral genome copies, and release of infectious virus. It remains unknown the contribution of spontaneous virus production to the expansion of KSHV-infected tumor cells and the cellular factors that render the lymphatic environment unique to KSHV life cycle. We show here that expansion of the infected cell population, observed in LECs, but not in blood endothelial cells, is dependent on the spontaneous virus production from infected LECs. The drivers of lymphatic endothelium development, SOX18 and PROX1, regulated different steps of the KSHV life cycle. SOX18 enhanced the number of intracellular viral genome copies and bound to the viral origins of replication. Genetic depletion or chemical inhibition of SOX18 caused a decrease of KSHV genome copy numbers. PROX1 interacted with ORF50, the viral initiator of lytic replication, and bound to the KSHV genome in the promoter region of ORF50, increasing its transactivation activity and KSHV spontaneous lytic gene expression and infectious virus release. In Kaposi sarcoma tumors, SOX18 and PROX1 expression correlated with latent and lytic KSHV protein expression. These results demonstrate the importance of two key transcriptional drivers of LEC fate in the regulation of the tumorigenic KSHV life cycle. Moreover, they introduce molecular targeting of SOX18 as a potential novel therapeutic avenue in Kaposi sarcoma.Significance: SOX18 and PROX1, central regulators of lymphatic development, are key factors for KSHV genome maintenance and lytic cycle in lymphatic endothelial cells, supporting Kaposi sarcoma tumorigenesis and representing attractive therapeutic targets