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 sub-types 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.

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 CEAexpressing 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.

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.

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IntroductionTumors 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.MethodsIn 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.ResultsThe 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.ConclusionsThese 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.

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.

The short half-life and use of recombinant bone morphogenetic protein (BMP)2 in large doses poses major limitations in the clinic. Events regulating posttranslational processing and degradation of BMP2 in situ, linked to its secretion, have not been understood. Toward 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 stability 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 that preventing intracellular proteasomal loss of BMP2 through genetic modifications can overcome limitations related to its short half-life. K E Y W O R D SBMP2, proteasomal degradation, protein turnover, ubiquitination | 181 KHATTAR eT Al.

We conclude that perturbing the Hsp90 pathway could provide a useful therapeutic strategy in tumors driven by Cks1 overexpression.