Faculty Bibliography

Ex vivo (EV)-derived megakaryocytes (MKs) have shown great promise as a substitute for platelets in transfusion medicine to alleviate a severe shortage of donor-platelets. Challenges remain that include poor efficiency, a limited scale of production, and undefined short-term storage conditions of EV-derived MKs. This study aims to develop a high-efficiency system for large-scale production of Good Manufacturing Practice (GMP)-grade MKs and determine the short-term storage condition for the MKs. A roller-bottle culture system was introduced to produce GMP-grade MKs from small-molecule/cytokine cocktail expanded hematopoietic stem cells. Various buffer systems and temperatures for the short-term storage of MKs were assessed by cell viability, biomarker expression, and DNA ploidy levels. MKs stored for 24 hours were transplanted into sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to confirm their platelet-releasing and tissue-homing ability in vivo. A yield of ~ 2.5 × 10⁴ CD41a⁺ /CD42b⁺ MKs with purity of ~ 80% was achieved from one original cord blood CD34⁺ cell. Compared with the static culture, the roller-bottle culture system significantly enhanced megakaryopoiesis, as shown by the cell size, DNA ploidy, and megakaryopoiesis-related gene expression. The optimal storage condition for the MKs was defined as normal saline with 10% human serum albumin at 22℃. Stored MKs were capable of rapidly producing functional platelets and largely distributing in the lungs of NOD/SCID mice. The novel development of efficient production and storage system for GMP-grade MKs represents a significant step toward application of these MKs in the clinic.

BACKGROUND:Hematopoietic stem cell (HSC) transplantation has been widely applied to the treatment of malignant blood diseases. However, limited number of functional HSCs hinders successful transplantation. The purpose of our current study is to develop a new and cost-efficient medium formulation that could greatly enhance the expansion of HSCs while retaining their long-term repopulation and hematopoietic properties for effective clinical transplantation. METHODS:cells were expanded with a new, cost-efficient expansion medium formulation, named hematopoietic expansion medium (HEM), consisting of various cytokines and nutritional supplements. The long-term repopulation potential and hematologic-lineage differentiation ability of expanded human cells were studied in the non-obese diabetic/severe combined immunodeficiency mouse model. Furthermore, the efficacy and safety studies were performed by autologous transplantation of expanded primate cells in the nonhuman primate model. RESULTS:cells survived for over 18 months without any noticeable abnormalities. CONCLUSIONS:Together, these findings demonstrate promising potential for the utility of HEM to improve expansion of HSCs for clinical application.

AhR is an environmental response gene that mediates cellular responses to a variety of xenobiotic compounds that frequently function as AhR ligands. Many AhR ligands are classified as carcinogens or pro-carcinogens. Thus, AhR itself acts as a major mediator of the carcinogenic effect of many xenobiotics in vivo. In this concise review, mechanisms by which AhR trans-activates downstream target gene expression, modulates immune responses, and mediates malignant transformation and tumor development are discussed. Moreover, activation of AhR by post-translational modifications and crosstalk with other transcription factors or signaling pathways are also summarized.

Over half a million US residents are suffering with bladder cancer (BC), which costs a total $4 billion in treatment annually. Although recent studies report that autophagy-related gene 7 (ATG7) is overexpressed in BCs, the regulatory effects of ATG7 on cancer stem-like phenotypes and invasion have not been explored yet. Current studies demonstrated that the deficiency of ATG7 by its shRNA dramatically reduced sphere formation and invasion in vitro, as well as lung metastasis in vivo in human invasive BC cells. Further studies indicated that the knockdown of ATG7 attenuated the expression of CD44 standard (CD44s), while ectopic introduction of CD44s, was capable of completely restoring sphere formation, invasion, and lung metastasis in T24T(shATG7) cells. Mechanistic studies revealed that ATG7 overexpression stabilized CD44s proteins accompanied with upregulating USP28 proteins. Upregulated USP28 was able to bind to CD44s and remove the ubiquitin group from CD44s' protein, resulting in the stabilization of CD44s protein. Moreover, ATG7 inhibition stabilized AUF1 protein and thereby reduced tet1 mRNA stability and expression, which was able to demethylate usp28 promoter, reduced USP28 expression, finally promoting CD44s degradation. In addition, CD44s was defined to inhibit degradation of RhoGDIβ, which in turn promotes BC invasion. Our results demonstrate that CD44s is a key ATG7 downstream regulator of the sphere formation, invasion, and lung metastasis of BCs, providing significant insight into understanding the BC invasions, metastasis, and stem-like properties.

Ras proteins participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, and differentiation. We have previously reported that Ras proteins are modified by sumoylation and that lysine-42 (K42) plays an important role in mediating the modification.  In the current study, we further investigated the role of K42 in regulating cellular activities of K-Ras. Inducible expression of K-RasV12 led to the activation of downstream components including c-RAF, MEK1, and ERKs whereas expression of K-RasV12/R42 mutant compromised the activation of the RAF/MEK/ERK signaling axis. Expression of K-RasV12/R42 also led to reduced phosphorylation of several other protein kinases including JNK, Chk2, and FAK. Significantly, K-RasV12/R42 expression inhibited cellular migration and invasion in vitro in multiple cell lines including transformed pancreatic cells. Given K-Ras plays a crucial role in mediating oncogenesis in pancreas, we treated transformed pancreatic cells of both BxPC-3 and MiaPaCa-2 with 2-D08, an SUMO E2 inhibitor. Treatment with the compound inhibited cell migration in a concentration-dependent manner, which was correlated with a reduced level of K-Ras sumoylation. Moreover, 2-D08 suppressed expression of ZEB1 (a mesenchymal cell marker) with concomitant induction of ZO-1 (an epithelial cell marker). Combined, our studies strongly suggest that post-translational modification(s) including sumoylation mediated by K42 plays a crucial role in K-Ras activities in vivo.

RAS proteins are GTPases that participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, differentiation, and autophagy. Mutations or deregulated activities of RAS are frequently the driving force for oncogenic transformation and tumorigenesis. Given the important roles of the small ubiquitin-related modifier (SUMO) pathway in controlling the stability, activity, or subcellular localization of key cellular regulators, we investigated here whether RAS proteins are posttranslationally modified (i.e. SUMOylated) by the SUMO pathway. We observed that all three RAS protein isoforms (HRAS, KRAS, and NRAS) were modified by the SUMO3 protein. SUMOylation of KRAS protein, either endogenous or ectopically expressed, was observed in multiple cell lines. The SUMO3 modification of KRAS proteins could be removed by SUMO1/sentrin-specific peptidase 1 (SENP1) and SENP2, but not by SENP6, indicating that RAS SUMOylation is a reversible process. A conserved residue in RAS, Lys-42, was a site that mediates SUMOylation. Results from biochemical and molecular studies indicated that the SUMO-E3 ligase PIASγ specifically interacts with RAS and promotes its SUMOylation. Moreover, SUMOylation of RAS appeared to be associated with its activation. In summary, our study reveals a new posttranslational modification for RAS proteins. Since we found that HRAS, KRAS, and NRAS can all be SUMOylated, we propose that SUMOylation might represent a mechanism by which RAS activities are controlled.

The cellular hypoxic response contributes to cell transformation and tumor progression. Hypoxia-inducible factor 1 (HIF-1) is a key transcription factor that mediates transcription of genes whose products are essential for cellular adaptation to hypoxia. The activity of HIF-1 is largely regulated by the abundance of its alpha subunit (HIF-1alpha), which is primarily regulated by an oxygen-dependent and ubiquitin/proteasome-mediated degradation process. The HIF-1alpha protein level is also regulated by protein kinases through phosphorylation. Polo-like kinase 3 (Plk3) is a serine/threonine protein kinase with a tumor suppressive function. Plk3 phosphorylates and destabilizes HIF-1alpha. Plk3 also phosphorylates and stabilizes PTEN, a known regulator of HIF-1alpha stability via the PI3K pathway. Our latest study showed that the Plk3 protein is suppressed by hypoxia or nickel treatment via the ubiquitin/proteasome system. We discovered that Seven in Absentia Homologue 2 (SIAH2) is the E3 ubiquitin ligase of Plk3 and that Plk3 in turn destabilizes SIAH2. Given the role of SIAH2 in promoting stability of HIF-1alpha, our work reveals a novel mutual regulatory mechanism between Plk3 and SIAH2, which may function to fine-tune the cellular hypoxic response. Here we discuss the role of Plk3 in the hypoxic response and tumorigenesis in light of these latest findings.

Conventional high-dose chemotherapy frequently leads to severe neutropenia, during which patients experience a high risk of infection. Although support care with donor's neutrophils is possible this choice is largely hampered by the limited availability of matched donors. To overcome this problem, we explored a large-scale ex vivo production of neutrophils from hematopoietic stem cells (HSCs) using a four-stage culture approach in a roller-bottle production platform. We expanded CD34+ HSCs isolated from umbilical cord blood (UCB) using our in-house special medium supplemented with cytokine cocktails and achieved about 49000-fold expansion of cells, among which about 61% were differentiated mature neutrophils. Ex vivo differentiated neutrophils exhibited a chemotactic activity similar to those from healthy donors and were capable of killing E. coli in vitro. The expansion yield as reported herein was at least 5 times higher than any other methods reported in the literature. Moreover, the cost of our modified medium was only a small fraction (<1/60) of the StemSpan SFEM. Therefore, our ex vivo expansion platform, coupled with a low cost of stem cell culture due to the use of a modified medium, makes large-scale manufacturing neutrophils possible, which should be able to greatly ameliorate neutrophil shortage for transfusion in the clinic.