Systemic Checkpoint Blockade by PD-L1 Single-Chain Antibody Confers Potent Antitumor Immunity and Long-term Survival.

Immune checkpoint inhibitors (ICI) are promising in adjuvant settings for solid tumors and hematologic malignancies. They are currently used in the treatment as mAbs in high concentrations, raising concerns of toxicity and adverse side effects. Among various checkpoint molecules, targeting the programmed cell death protein-1 (PD-1)-programmed death-ligand 1 (PD-L1) axis has garnered more clinical utility than others have. To develop a physiologically relevant and systemically stable level of ICIs from a one-time application by genetic antibody engineering, we endeavored using a nonpathogenic, replication-deficient recombinant adeno-associated vector (rAAV) expressing single-chain variable fragments (scFv) of PD-L1 antibody and tested in syngeneic mouse therapy models of MC38 colorectal and EMT6 breast tumors. Results of this study indicated a significant protection against PD-L1-mediated inhibition of CD8+ T-cell function, against the growth of primary and secondary tumors, and durable antitumor CTLs activity by adoptive CD8+ T-cell transfer. Stable maintenance of PD-L1 scFv in vivo resulted in an increase in PD-1- CD8+ T cells and a concomitant decrease in regulatory T cells, M2 macrophages, and myeloid-derived suppressor cells in the tumor microenvironment. Overall, these data demonstrate the potential of rAAV-PD-L1-scFv as an alternative to mAb targeting of PD-L1 for tumor therapy.

Calcium selective channel TRPV6: Structure, function, and implications in health and disease.

Calcium-selective channel TRPV6 (Transient Receptor Potential channel family, Vanilloid subfamily member 6) belongs to the TRP family of cation channels and plays critical roles in transcellular calcium (Ca2+) transport, reuptake of Ca2+ into cells, and maintaining a local low Ca2+ environment for certain biological processes. Recent crystal and cryo-electron microscopy-based structures of TRPV6 have revealed mechanistic insights on how the protein achieves Ca2+ selectivity, permeation, and inactivation by calmodulin. The TRPV6 protein is expressed in a range of epithelial tissues such as the intestine, kidney, placenta, epididymis, and exocrine glands such as the pancreas, prostate and salivary, sweat, and mammary glands. The TRPV6 gene is a direct transcriptional target of the active form of vitamin D and is efficiently regulated to meet the body's need for Ca2+ demand. In addition, TRPV6 is also regulated by the level of dietary Ca2+ and under physiological conditions such as pregnancy and lactation. Genetic models of loss of function in TRPV6 display hypercalciuria, decreased bone marrow density, deficient weight gain, reduced fertility, and in some cases alopecia. The models also reveal that the channel plays an indispensable role in maintaining maternal-fetal Ca2+ transport and low Ca2+ environment in the epididymal lumen that is critical for male fertility. Most recently, loss of function mutations in TRPV6 gene is linked to transient neonatal hyperparathyroidism and early onset chronic pancreatitis. TRPV6 is overexpressed in a wide range of human malignancies and its upregulation is strongly correlated to tumor aggressiveness, metastasis, and poor survival in selected cancers. This review summarizes the current state of knowledge on the expression, structure, biophysical properties, function, polymorphisms, and regulation of TRPV6. The aberrant expression, polymorphisms, and dysfunction of this protein linked to human diseases are also discussed.

RANKL-Targeted Combination Therapy with Osteoprotegerin Variant Devoid of TRAIL Binding Exerts Biphasic Effects on Skeletal Remodeling and Antitumor Immunity.

Complexities in treating breast cancer with bone metastasis are enhanced by a vicious protumorigenic pathology, involving a shift in skeletal homeostasis toward aggressive osteoclast activity and polarization of immune cells supporting tumor growth and immunosuppression. Recent studies signify the role of receptor activator of NF-κB ligand (RANKL) beyond skeletal pathology in breast cancer, including tumor growth and immunosuppression. By using an osteoprotegerin (OPG) variant, which we developed recently through protein engineering to uncouple TNF-related apoptosis-inducing ligand (TRAIL) binding, this study established the potential of a cell-based OPGY49R therapy for both bone damage and immunosuppression in an immunocompetent mouse model of orthotopic and metastatic breast cancers. In combination with agonistic death receptor (DR5) activation, the OPGY49R therapy significantly increased both bone remolding and long-term antitumor immunity, protecting mice from breast cancer relapse and osteolytic pathology. With limitations, cost, and toxicity issues associated with the use of denosumab, bisphosphonates, and chemotherapy for bone metastatic disease, use of OPGY49R combination could offer a viable alternate therapeutic approach.

Structural determinants and genetic modifications enhance BMP2 stability and extracellular secretion.

The short half-life and use of recombinant bone morphogenetic protein (BMP)-2 in large doses poses major limitations in the clinic. Events regulating post-translational processing and degradation of BMP2 in situ, linked to its secretion, have not been understood. Towards identifying mechanisms regulating intracellular BMP2 stability, we first discovered that inhibiting proteasomal degradation enhances both intracellular BMP2 level and its extracellular secretion. Next, we identified BMP2 degradation occurs through an ubiquitin-mediated mechanism. Since ubiquitination precedes proteasomal turnover and mainly occurs on lysine residues of nascent proteins, we systematically mutated individual lysine residues within BMP2 and tested them for enhanced stability. Results revealed that substitutions on four lysine residues within the pro-BMP2 region and three in the mature region increased both BMP2 turnover and extracellular secretion. Structural modeling revealed key lysine residues involved in proteasomal degradation occupy a lysine cluster near proprotein convertase cleavage site. Interestingly, mutations within these residues did not affect biological activity of BMP2. These data suggest preventing intracellular proteasomal loss of BMP2 through genetic modifications can overcome limitations related to its short half-life.

Recombinant AAV-CEA Tumor Vaccine in Combination with an Immune Adjuvant Breaks Tolerance and Provides Protective Immunity.

Carcinoembryonic antigen (CEA) is a human glycoprotein involved in cellular adhesion and expressed during human fetal development. Although expression of CEA largely ceases prior to birth, several human epithelial cancers, including colorectal, gastric, squamous esophageal, and breast carcinomas have been known to overexpress CEA, suggesting its potential as an immunotherapeutic target. Using a transgenic mouse model constitutively expressing human CEA in a spatiotemporal manner as a self-protein and a syngeneic mouse colon cancer cell line, MC38-CEA, overexpressing CEA, we tested the potential of a novel genetic immunotherapy approach against CEA-expressing tumors, using recombinant adeno-associated virus vector encoding CEA (rAAV-CEA) and appropriately timed immune adjuvant application. Results of the study demonstrated breaking of immune tolerance for CEA with this vaccine regimen and an anti-tumor response, resulting in tumor-free survival. Furthermore, tumor challenge of CEA-vaccinated mice with parental MC38 cells not expressing CEA did not result in protection from tumor development, confirming that the protection against tumor development is CEA specific. The study illustrates the feasibility of utilizing rAAV vectors in combination with an immunostimulatory adjuvant to break tolerance to weakly immunogenic self-antigens and for an anti-tumor response.

Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations.

The lack of consensus on bone marrow (BM) and splenic immune cell profiles in preclinical mouse strains complicates comparative analysis across different studies. Although studies have documented relative distribution of immune cells from peripheral blood in mice, similar studies for BM and spleen from naïve mice are lacking. In an effort to establish strain- and gender-specific benchmarks for distribution of various immune cell subtypes in these organs, we performed immunophenotypic analysis of BM cells and splenocytes from both genders of three commonly used murine strains (C57BL/6NCr, 129/SvHsd, and BALB/cAnNCr). Total neutrophils and splenic macrophages were significantly higher in C57BL/6NCr, whereas total B cells were lower. Within C57BL/6NCr female mice, BM B cells were elevated with respect to the males whereas splenic mDCs and splenic neutrophils were reduced. Within BALB/cAnNCr male mice, BM CD4+ Tregs were elevated with respect to the other strains. Furthermore, in male BALB/cAnNCr mice, NK cells were elevated with respect to the other strains in both BM and spleen. Splenic CD4+ Tregs and splenic CD8+ T cells were reduced in male BALB/c mice in comparison to female mice. Bone marrow CD4+ T cells and mDCs were significantly increased in 129/SvHsd whereas splenic CD8+ T cells were reduced. In general, males exhibited higher immature myeloid cells, macrophages, and NK cells. To our knowledge, this study provides a first attempt to systematically establish organ-specific benchmarks on immune cells in studies involving these mouse strains.

Location of tumor affects local and distant immune cell type and number.

INTRODUCTION: Tumors comprise heterogeneous populations of cells, including immune infiltrates that polarize during growth and metastasis. Our preclinical studies on breast cancer (BCa) identified functional differences in myeloid-derived suppressor cells based on tumor microenvironment (TME), prompting variations in host immune response to tumor growth, and dissemination based on tissue type. METHODS: In order to understand if such variations existed among other immune cells, and if such alteration occurs in response to tumor growth at the primary site or due to bone dissemination, we characterized immune cells, examining localized growth and in the tibia. In addition, immune cells from the spleen were examined from animals of both tumor locations by flow cytometry. RESULTS: The study demonstrates that location of tumor, and not simply the tumor itself, has a definitive role in regulating immune effectors. Among all immune cells characterized, macrophages were decreased and myeloid dendritic cell were increased in both tumor locations. This difference was more evident in subcutaneous tumors. Additionally, spleens from mice with subcutaneous tumors contained greater increases in both macrophages and myeloid dendritic cells than in mice with bone tumors. Furthermore, in subcutaneous tumors there was an increase in CD4+ and CD8+ T-cell numbers, which was also observed in their spleens. CONCLUSIONS: These data indicate that alterations in tumor-reactive immune cells are more pronounced at the primary site, and exert a similar change at the major secondary lymphoid organ than in the bone TME. These findings could provide translational insight into designing therapeutic strategies that account for location of metastatic foci.

Cks1 proteasomal turnover is a predominant mode of regulation in breast cancer cells: role of key tyrosines and lysines.

Constitutive levels of Cks1 protein are very high in mammary carcinoma tissue and in breast tumor cell lines. However, despite being transcribed at relatively high levels, Cks1 protein is very low in normal mammary tissue. Also, basal Cks1 is barely detectable in primary human mammary epithelial cells (HMECs). Epoximicin, a proteasome inhibitor, induced detectable endogenous Cks1 in HMECs, and upregulated it above the basal level in MCF-7 breast cancer cells. Interestingly, transiently transfected Cks1 is remarkably unstable and accumulates only upon proteasomal blockade in multiple cell lines even when driven by the strong CMV promoter-enhancer. We examined the stability of site-directed Cks1 mutants in order to identify the structural determinants of its turnover in cancer cells. Since protein turnover is regulated by phosphorylation, and phosphoproteomic studies reveal phosphorylated tyrosines in Cks1, we replaced its five conserved tyrosines (Y) with phenylalanine (F), both individually and in combinations. We find that like wild-type, all transiently transfected mutant Cks1 vectors, even when driven by the CMV promoter-enhancer, expressed detectable protein only in cells treated with epoximicin. However, turnover of the Y8F, Y12F and Y19F Cks1 mutants was more rapid than that of wild-type, Y7F and Y57F. Since lysines are modified by ubiquitination or acetylation we also examined the consequences of lysine to arginine (K-R) substitutions on Cks1 proteasomal turnover. We found that the individual mutations K4R, K26R, K30R, and K34R slowed Cks1 turnover, while the K79R mutation or the combined mutation K75-76-78-79R increased turnover. Taken together, regulation of Cks1 protein stability is crucially dependent on specific tyrosine and lysine residues which are potential sites for post-translational modifications.

Cks1 proteasomal degradation is induced by inhibiting Hsp90-mediated chaperoning in cancer cells.

PURPOSE: Cks1, a conformationally heterogenous 9 kDa protein, is markedly overexpressed in cancer cells and contributes to tumor development. Cks1 is an essential component of the SCF-Skp2 ubiquitin ligase complex that targets the Cdk inhibitors p27(Kip1) and p21(Cip1). Cks1 is known to interact with the Hsp90-Cdc37 chaperone machinery, although whether this facilitates its conformational maturation and stability is not known. To test whether abrogating the chaperone function of Hsp90 could destabilize Cks1, we examined the effects of treating different cancer cell lines with the benzoquinone ansamycin 17-allylamino geldanamycin (17-AAG), a compound that selectively binds Hsp90 and potently inhibits its ATP-dependent chaperone activity. METHODS: The effect of Hsp90 inhibition using 17-AAG on Cks1 protein and associated cell cycle proteins including Skp2, p27(Kip1), p21(Cip1), and Cdk1 in cancer cells was determined by Western blotting. Ubiquitination analysis was carried out by transfecting cells with an HA-ubiquitin plasmid and specifically immunoprecipitating Cks1 to examine polyubiquitinated species. Flow cytometry was utilized to examine the effects of Hsp90 inhibition on cell cycle profiles. RESULTS: Here, we demonstrate for the first time that inhibition of Hsp90 utilizing 17-AAG destabilizes Cks1 in cancer cells by promoting its ubiquitination and proteasomal degradation. 17-AAG-induced Cks1 depletion was accompanied by concomitant decreases in Skp2 and Cdk1. 17-AAG treatment also induced G2/M accumulation in MCF-7 breast carcinoma cells, and G1 accumulation in the colon carcinoma lines HCT116 and SW620. CONCLUSIONS: We conclude that perturbing the Hsp90 pathway could provide a useful therapeutic strategy in tumors driven by Cks1 overexpression.

Cks1: Structure, Emerging Roles and Implications in Multiple Cancers.

Deregulation of the cell cycle results in loss of normal control mechanisms that prevent aberrant cell proliferation and cancer progression. Regulation of the cell cycle is a highly complex process with many layers of control. One of these mechanisms involves timely degradation of CDK inhibitors (CKIs) like p27Kip1 by the ubiquitin proteasomal system (UPS). Cks1 is a 9 kDa protein which is frequently overexpressed in different tumor subtypes, and has pleiotropic roles in cell cycle progression, many of which remain to be fully characterized. One well characterized molecular role of Cks1 is that of an essential adaptor that regulates p27Kip1 abundance by facilitating its interaction with the SCF-Skp2 E3 ligase which appends ubiquitin to p27Kip1 and targets it for degradation through the UPS. In addition, emerging research has uncovered p27Kip1-independent roles of Cks1 which have provided crucial insights into how it may be involved in cancer progression. We review here the structural features of Cks1 and their functional implications, and also some recently identified Cks1 roles and their involvement in breast and other cancers.