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This research was funded in part by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K., an NIH/NICHD grant (RO1 HD-07857) to B.W.O.M., and a NIH/NICHD grant (R01 HD-042311) to J.P.L. The authors declare no conflicts of interests.

Although endometrial cancer is the most common cancer of the female reproductive tract, we have little understanding of what controls endometrial cancer beyond the transcriptional effects of steroid hormones such as estrogen. As a result, we have limited therapeutic options for the ~62,000 women diagnosed with endometrial cancer each year in the United States. Here, in an attempt to identify new prognostic and therapeutic targets, we focused on a new area for this cancer—alternative mRNA splicing—and investigated whether splicing factor, SF3B1, plays an important role in endometrial cancer pathogenesis. Using a tissue microarray, we found that human endometrial tumors expressed more SF3B1 protein than non-cancerous tissues. Furthermore, SF3B1 knockdown reduced in vitro proliferation, migration, and invasion of the endometrial cancer cell lines Ishikawa and AN3CA. Similarly, the SF3B1 inhibitor, Pladienolide-B (PLAD-B), reduced the Ishikawa and AN3CA cell proliferation and invasion in vitro. Moreover, PLAD-B reduced tumor growth in an orthotopic endometrial cancer mouse model. Using RNA-Seq approach, we identified ~2000 differentially expressed genes (DEGs) with SF3B1 knockdown in endometrial cancer cells. Additionally, alternative splicing (AS) events analysis revealed that SF3B1 depletion led to alteration in multiple categories of AS events including alternative exon skipping (ES), transcript start site usage (TSS), and transcript termination site (TTS) usage. Subsequently, bioinformatics analysis showed KSR2 as a potential candidate for SF3B1-mediated functions in endometrial cancer. Specifically, loss of SF3B1 led to decrease in KSR2 expression, owing to reduced maturation of KSR2 pre-mRNA to a mature RNA. Importantly, we found rescuing the KSR2 expression with SF3B1 knockdown partially restored the cell growth of endometrial cancer cells. Taken together, our data suggest that SF3B1 plays a crucial oncogenic role in the tumorigenesis of endometrial cancer and hence may support the development of SF3B1 inhibitors to treat this disease.

The coronavirus disease 2019 (COVID-19) first appeared in December 2019 and rapidly spread throughout the world. The SARS-CoV-2 virus enters the host cells by binding to the angiotensin-converting enzyme (ACE2). Although much of the focus is on respiratory symptoms, recent reports suggest that SARS-CoV-2 can cause pregnancy complications such as pre-term birth and miscarriages; and women with COVID-19 had maternal vascular malperfusion and decidual arteriopathy in their placentas. Here, we report that ACE2 protein is expressed in both endometrial epithelial and stromal cells in the proliferative phase of the menstrual cycle, and expression increases in stromal cells in the secretory phase. The ACE2 mRNA and protein abundance increased during primary human endometrial stromal cell (HESC) decidualization. Further, HESCs transfected with ACE2-targeting siRNA impaired the full decidualization response, as evidenced by a lack of morphology change and lower expression of the decidualization markers PRL and IGFBP1. Additionally, in mice during pregnancy, ACE2 protein was expressed in uterine epithelial, and stromal cells increased through day six of pregnancy. Finally, progesterone induced Ace2 mRNA expression in mouse uteri more than vehicle or estrogen. These data establish a role for ACE2 in endometrial physiology, suggesting that SARS-CoV-2 may be able to enter endometrial stromal cells and elicit pathological manifestations in women with COVID-19 including an increased risk of early pregnancy loss.

Transforming growth factor-beta (TGF-B) plays an important role in embryo implantation; however, TGF-B requires liberation from its inactive latent forms (i.e., large latent TGF-B complex [LLC] and small latent TGF-B complex [SLC]) to its biologically active (i.e., monomer or dimer) forms in order to act on its receptors (TGF-BRs), which in turn activate SMAD2/3. Activation of TGF-B1 from its latent complexes in the uterus is not yet deciphered. We investigated uterine latent TGF-B1 complex and its biologically active form during implantation, decidualization, and delayed implantation. Our study, utilizing nonreducing SDS-PAGE followed by Western blotting and immunoblotting with TGF-B1, LTBP1, and latency-associated peptide, showed the presence of LLC and SLC in the uterine extracellular matrix and plasma membranous protein fraction during stages of the implantation period. A biologically active form of TGF-B1 (~17-kDa monomer) was highly elevated in the uterine plasma membranous compartment at the peri-implantation stage (implantation and nonimplantation sites). Administration of hydroxychloroquine (an inhibitor of pro-TGF-B processing) at the preimplantation stage was able to block the liberation of biologically active TGF-B1 from its latent complex at the postimplantation stage; as a consequence, the number of implantation sites was reduced at Day 5 (1000 h), as was the number of fetuses at Day 13. The inhibition of TGF-B1 showed reduced levels of phosphorylated SMAD3. Further, the delayed-implantation mouse model showed progesterone and estradiol coordination to release the active TGF-B1 form from its latent complex in the receptive endometrium. This study demonstrates the importance of liberation of biologically active TGF-B1 during the implantation period and its regulation by estradiol.