So Youn Kim

Toward precision medicine for preserving fertility in cancer patients: existing and emerging fertility preservation options for women

As the number of young cancer survivors increases, quality of life after cancer treatment is becoming an ever more important consideration. According to a report from the American Cancer Society, approximately 810,170 women were diagnosed with cancer in 2015 in the United States. Among female cancer survivors, 1 in 250 are of reproductive age. Anticancer therapies can result in infertility or sterility and can have long-term negative effects on bone health, cardiovascular health as a result of reproductive endocrine function. Fertility preservation has been identified by many young patients diagnosed with cancer as second only to survival in terms of importance. The development of fertility preservation technologies aims to help patients diagnosed with cancer to preserve or protect their fertility prior to exposure to chemo- or radiation therapy, thus improving their chances of having a family and enhancing their quality of life as a cancer survivor. Currently, sperm, egg, and embryo banking are standard of care for preserving fertility for reproductive-age cancer patients; ovarian tissue cryopreservation is still considered experimental. Adoption and surrogate may also need to be considered. All patients should receive information about the fertility risks associated with their cancer treatment and the fertility preservation options available in a timely manner, whether or not they decide to ultimately pursue fertility preservation. Because of the ever expanding number of options for treating cancer and preserving fertility, there is now an opportunity to take a precision medicine approach to informing patients about the fertility risks associated with their cancer treatment and the fertility preservation options that are available to them.

Patents on SELEX and Therapeutic Aptamers

Aptamers, the oligonucleotides (DNA/RNA) that bind to target molecules with high specificity and affinity, have been a focus of therapeutic research for the last two decades. The magnitude of scientific and commercial interest shown for aptamers is not surprising because aptamers have several advantages over other curative modalities, especially antibodies. Patent activity in this field has also shown an exponential growth. Aptamers against a broad range of disease-causing pathogens and proteins have been patented. These have potential use as a biomarker, therapeutics and diagnostics. As drugs they have shown commendable results in cell and animal models, a few of them undergoing clinical trials. In this review, we discuss upon all important patents filed on therapeutic aptamers and SELEX technology employed to synthesize them. We have classified them in categories based upon their target or the diseased condition they apt for. These patents provide insight into the development that occurred in transformation of aptamers as therapeutic entities and reinforces the potential they have.

Alkaline Phosphatase ALPPL-2 Is a Novel Pancreatic Carcinoma-Associated Protein

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a very low median survival rate. The lack of early sensitive diagnostic markers is one of the main causes of PDAC-associated lethality. Therefore, to identify novel pancreatic cancer biomarkers that can facilitate early diagnosis and also help in the development of effective therapeutics, we developed RNA aptamers targeting pancreatic cancer by Cell-systematic evolution of ligands by exponential enrichment (SELEX) approach. Using a selection strategy that could generate aptamers for 2 pancreatic cancer cell lines in one selection scheme, we identified an aptamer SQ-2 that could recognize pancreatic cancer cells with high specificity. Next, by applying 2 alternative approaches: (i) aptamer-based target pull-down and (ii) genome-wide microarray-based identification of differentially expressed mRNAs in aptamer-positive and -negative cells, we identified alkaline phosphatase placental-like 2 (ALPPL-2), an oncofetal protein, as the target of SQ-2. ALPPL-2 was found to be ectopically expressed in many pancreatic cancer cell lines at both mRNA and protein levels. RNA interference-mediated ALPPL-2 knockdown identified novel tumor-associated functions of this protein in pancreatic cancer cell growth and invasion. In addition, the aptamer-mediated identification of ALPPL-2 on the cell surface and cell secretions of pancreatic cancer cells supports its potential use in the serum- and membrane-based diagnosis of PDAC.

Foxl2, a Forkhead Transcription Factor, Modulates Nonclassical Activity of the Estrogen Receptor-α

Foxl2 is a forkhead transcription factor required for ovary development and ovarian follicle maturation. In this report, we identified and characterized a functional relationship between Foxl2 expression and estrogen receptor (ER)-alpha signaling. We show that Foxl2 has no effect on classical ERalpha-mediated transcription, which occurs through canonical estrogen response elements. However, Foxl2 suppresses ERalpha signaling through nonclassical tethered transcriptional pathways. Specifically, the selective ER modulator tamoxifen stimulates activator protein-1 (AP1)-dependent transcription via the ERalpha, and this enhancement is blocked by Foxl2. Two lines of evidence suggest that Foxl2 suppression is mediated by physical interactions with ERalpha rather than direct action at AP1 binding sites. First, ERalpha is coimmunoprecipitated with Foxl2. Second, activation of a upstream activating sequence (UAS) reporter by Gal4-cJun in the presence of ERalpha and tamoxifen was blocked by Foxl2, demonstrating suppression in the absence of an AP1 site. Cyclooxygenase-2 (COX2), which is required for ovulation, was identified through expression profiling as a candidate physiological target for nonclassical ERalpha signaling and thus modulation by ERalpha/Foxl2 interactions. This possibility was confirmed by two sets of experiments. COX2 protein levels were induced by ERalpha in the presence of tamoxifen, and protein expression was suppressed by Foxl2. In addition, ERalpha stimulation of the COX2 promoter was repressed by Foxl2. We conclude that ERalpha and Foxl2 interact and that Foxl2 selectively suppresses ERalpha-mediated transcription of AP1-regulated genes. These data provide a potential point of convergence for ERalpha and Foxl2 to regulate ovarian development and function.

Inflexinol inhibits colon cancer cell growth through inhibition of nuclear factor-κB activity via direct interaction with p50

Kaurane diterpene compounds have been known to be cytotoxic against several cancer cells through inhibition of nuclear factor-κB (NF-κB) activity. Here, we showed that inflexinol, a novel kaurane diterpene compound, inhibited the activity of NF-κB and its target gene expression as well as cancer cell growth through induction of apoptotic cell death in vitro and in vivo. These inhibitory effects on NF-κB activity and on cancer cell growth were suppressed by the reducing agents DTT and glutathione and were abrogated in the cells transfected with mutant p50 (C62S). Sol-gel biochip and surface plasmon resonance analysis showed that inflexinol binds to the p50 subunit of NF-κB. These results suggest that inflexinol inhibits colon cancer cell growth via induction of apoptotic cell death through inactivation of NF-κB by a direct modification of cysteine residue in the p50 subunit of NF-κB. [Mol Cancer Ther 2009;8(6):1613–24]

Cell Autonomous Phosphoinositide 3-Kinase Activation in Oocytes Disrupts Normal Ovarian Function Through Promoting Survival and Overgrowth of Ovarian Follicles

In this study, we explored the effects of oocytic phosphoinositide 3-kinase (PI3K) activation on folliculogensis by generating transgenic mice, in which the oocyte-specific Cre-recombinase induces the expression of constitutively active mutant PI3K during the formation of primordial follicles. The ovaries of neonatal transgenic (Cre+) mice showed significantly reduced apoptosis in follicles, which resulted in an excess number of follicles per ovary. Thus, the elevation of phosphatidylinositol (3,4,5)-trisphosphate levels within oocytes promotes the survival of follicles during neonatal development. Despite the increase in AKT phosphorylation, primordial follicles in neonatal Cre+ mice remained dormant demonstrating a nuclear accumulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These primordial follicles containing a high level of nuclear PTEN persisted in postpubertal females, suggesting that PTEN is the dominant factor in the maintenance of female reproductive lifespan through the regulation of primordial follicle recruitment. Although the oocytic PI3K activity and PTEN levels were elevated, the activation of primordial follicles and the subsequent accumulation of antral follicles with developmentally competent oocytes progressed normally in prepubertal Cre+ mice. However, mature Cre+ female mice were anovulatory. Because postnatal day 50 Cre+ mice released cumulus-oocyte complexes with developmentally competent oocytes in response to super-ovulation treatment, the anovulatory phenotype was not due to follicular defects but rather endocrine abnormalities, which were likely caused by the excess number of overgrown follicles. Our current study has elucidated the critical role of oocytic PI3K activity in follicular function, as well as the presence of a PTEN-mediated mechanism in the prevention of immature follicle activation.

Long dsRNA-Mediated RNA Interference and Immunostimulation: A Targeted Delivery Approach Using Polyethyleneimine Based Nano-Carriers

RNA oligonucleotides capable of inducing controlled immunostimulation combined with specific oncogene silencing via an RNA interference (RNAi) mechanism provide synergistic inhibition of cancer cell growth. With this concept, we previously designed a potent immunostimulatory long double stranded RNA, referred to as liRNA, capable of executing RNAi mediated specific target gene silencing. In this study, we developed a highly effective liRNA based targeted delivery system to apply in the treatment of glioblastoma multiforme. A stable nanocomplex was fabricated by complexing multimerized liRNA structures with cross-linked branched poly(ethylene imine) (bPEI) via electrostatic interactions. We show clear evidence that the cross-linked bPEI was quite effective in enhancing the cellular uptake of liRNA on U87MG cells. Moreover, the liRNA-PEI nanocomplex provided strong RNAi mediated target gene silencing compared to that of the conventional siRNA-PEI complex. Further, the bPEI modification strategy with specific ligand attachment assisted the uptake of the liRNA-PEI complex on the mouse brain endothelial cell line (b.End3). Such delivery systems combining the beneficial elements of targeted delivery, controlled immunostimulation, and RNAi mediated target silencing have immense potential in anticancer therapy.

Target Gene Abundance Contributes to the Efficiency of siRNA-Mediated Gene Silencing

The gene-silencing activity of a small interfering RNA (siRNA) is determined by various factors. Considering that RNA interference (RNAi) is an unparalleled technology in both basic research and therapeutic applications, thorough understanding of the factors determining RNAi activity is critical. This report presents observations that siRNAs targeting KRT7 show cell-line-dependent activity, which correlates with the expression level of KRT7 mRNA. By modulating the target mRNA level, it was confirmed that highly expressed genes are more susceptible to siRNA-mediated gene silencing. Finally, several genes that show different expression levels in a cell-line dependent manner were tested, which verified the expression-level-dependent siRNA activities. These results strongly suggest that the abundance of target mRNA is a critical factor that determines the efficiency of the siRNA-mediated gene silencing in a given cellular context. This report should provide practical guidelines for designing RNAi experiments and for selecting targetable genes in RNAi therapeutics studies.

Constitutive Activation of PI3K in Oocyte Induces Ovarian Granulosa Cell Tumors.

Cell-cell interactions play crucial roles in the maintenance of tissue homeostasis, a loss of which often leads to varying diseases, including cancer. Here, we report that uncontrolled PI3K activity within oocytes irreversibly transforms granulosa cells (GC), causing GC tumors (GCT) through perturbed local cell communication. Previously, we reported reproductive phenotypes of transgenic mice, in which expression of constitutively active mutant PI3K was induced in primordial oocytes by Gdf9-iCre. The transgenic mice (Cre(+)) demonstrated severe ovarian phenotypes, including the overgrowth of excess ovarian follicles and anovulation. Surprisingly, the Cre(+) mice became cachectic by postnatal day 80 due to bilateral GCT. Although GCT cells proliferated independently of oocytes, local interactions with mutant PI3K-positive oocytes during early folliculogenesis were essential for the GC transformation. Growing GCT cells expressed high levels of inhibin βA and nuclear SMAD3, and the proliferation rate was positively correlated with a high activin A to inhibin A ratio. These results suggested that the tumor cells stimulated their growth through an activin A autocrine signaling pathway, a hypothesis confirmed by activin A secretion in cultured GCT cells, which proliferated in response. Although communication between the oocyte and surrounding somatic cells is critical for the normal development of ovarian follicles, perturbations in oocyte-GC communication during early folliculogenesis can induce GCT by activating an autocrine growth circuit program in GC. Cancer Res; 76(13); 3851-61. ©2016 AACR.

Geography of Follicle Formation in the Embryonic Mouse Ovary Impacts Activation Pattern during the First Wave of Folliculogenesis

ABSTRACT During embryonic development, mouse female germ cells enter meiosis in an anterior-to-posterior wave believed to be driven by retinoic acid. It has been proposed that ovarian follicle formation and activation follow the same general wave of meiotic progression; however, the precise anatomic specification of these processes has not been delineated. Here, we created a mouse line using Mvh, Gdf9, and Zp3 promoters to drive distinct temporal expression of three fluorescent proteins in the oocytes and to identify where the first follicle cohort develops. The fluorescent profile revealed that the first growing follicles consistently appeared in a specific region of the ovary, the anterior-dorsal region, which led us to analyze if meiotic onset occurred earlier in the dorsal ovarian region. Surprisingly, in addition to the anterior-to-posterior wave, we observed an early meiotic entry in the ventral region of the ovary. This additional anatomic stratification of meiosis contrasts with the localization of the initial follicle formation and activation in the dorsal region of the ovary. Therefore, our study suggests that the specification of cortical and medullar areas in the ventral and dorsal regions on the ovary, rather than the onset of meiosis, impacts where the first follicle activation event occurs.