Targeted pharmacologic inhibition of S-phase kinase-associated protein 2 (SKP2) mediated cell cycle regulation in lung and other RB-Related cancers: A brief review of current status and future prospects.

Small cell lung cancer (SCLC) often exhibits Rb deficiency, TRß and p130 deletion, and SKP2 amplification, suggesting TRß inactivation and SKP2 activation. It is reported that SKP2 targeted therapy is effective in some cancers in vitro and in vivo, but it is not reported for the treatment of SCLC and retinoblastoma. SKP2 is the synthetic lethal gene in SCLC and retinoblastoma, so SKP2 can be used for targeted therapy in SCLC and retinoblastoma. RB1 knockout mice develop several kinds of tumors, but Rb1 and SKP2 double knockout mice are healthy, suggesting that SKP2 targeted therapy may have significant effects on Rb deficient cancers with less side effects, and if successful in SCLC and retinoblastoma in vitro and in animal model, such compounds may be promising for the clinical treatment of SCLC, retinoblastoma, and variety of Rb deficient cancers. Previously our studies showed that retinoblastomas exhibit retinal cone precursor properties and depend on cone-specific thyroid hormone receptor ß2 (TRß2) and SKP2 signaling. In this study, we sought to suppress SCLC and retinoblastoma cell growth by SKP2 inhibitors as a prelude to targeted therapy in vitro and in vivo. We knocked down TRß2 and SKP2 or over-expressed p27 in SCLC and retinoblastoma cell lines to investigate SKP2 and p27 signaling alterations. The SCLC cell lines H209 as well as retinoblastoma cell lines Y79, WERI, and RB177 were treated with SKP2 inhibitor C1 at different concentrations, following which Western blotting, Immunostaining, and cell cycle kinetics studies were performed to study SKP2 and p27 expression ubiquitination, to determine impact on cell cycle regulation and growth inhibition. TRß2 knockdown in Y79, RB177 and H209 caused SKP2 downregulation and degradation, p27 up-regulation, and S phase arrest, whereas, SKP2 knockdown or p27 over-expression caused p27 accumulation and G1-S phase arrest. In the cell lines Y79, WERI, RB177, and H209 treatment with C1 caused SKP2 ubiquitination and degradation, p27 de-ubiquitination and accumulation, and cell growth arrest. SKP2 inhibitor C1 significantly suppressed retinoblastoma as well as SCLC cell growth by SKP2 degradation and p27 accumulation. In vivo study also showed inhibition of tumor growth with C1 treatment. Potential limitations of the success of such a therapeutic approach and its translational application in human primary tumors, and alternative approaches to overcome such limitations are briefly discussed for the treatment of retinoblastoma, SCLC and other RB-related cancers.

SKP2 Activation by Thyroid Hormone Receptor ß2 Bypasses Rb-Dependent Proliferation in Rb-Deficient Cells.

Germline RB1 mutations strongly predispose humans to cone precursor-derived retinoblastomas and strongly predispose mice to pituitary tumors, yet shared cell type-specific circuitry that sensitizes these different cell types to the loss of RB1 has not been defined. Here we show that the cell type-restricted thyroid hormone receptor isoform TRß2 sensitizes to RB1 loss in both settings by antagonizing the widely expressed and tumor-suppressive TRß1. TRß2 promoted expression of the E3 ubiquitin ligase SKP2, a critical factor for RB1-mutant tumors, by enabling EMI1/FBXO5-dependent inhibition of SKP2 degradation. In RB1 wild-type neuroblastoma cells, endogenous Rb or ectopic TRß2 was required to sustain SKP2 expression as well as cell viability and proliferation. These results suggest that in certain contexts, Rb loss enables TRß1-dependent suppression of SKP2 as a safeguard against RB1-deficient tumorigenesis. TRß2 counteracts TRß1, thus disrupting this safeguard and promoting development of RB1-deficient malignancies. Cancer Res; 77(24); 6838-50. ©2017 AACR.

Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2.

Chimeric antigen receptors (CAR) and bispecific antibodies (BsAb) are two powerful immunotherapy approaches for retargeting lymphocytes toward cancer cells. Despite their success in lymphoblastic leukemia, solid tumors have been more recalcitrant. Identifying therapeutic barriers facing CAR-modified (CART) or BsAb-redirected T (BsAb-T) cells should facilitate their clinical translation to solid tumors. Novel lentiviral vectors containing low-affinity or high-affinity 4-1BB second-generation anti-GD2 (disialoganglioside) CARs were built to achieve efficient T cell transduction. The humanized anti-GD2 × CD3 BsAb using the IgG-scFv platform was described previously. CART and BsAb-engaged T cells were tested for viability, proliferation, and activation/exhaustion marker expression, and in vitro cytotoxicity against GD2(+) tumor cells. The antitumor effect of CAR-grafted and BsAb-T cells was compared in a human melanoma xenograft model. The majority of high CAR density T cells were depleted upon exposure to GD2(+) target cells while the BsAb-T cells survived. The in vitro cytotoxicity of the surviving CART cells was inferior to that of the BsAb-T cells. Using low-affinity CARs, inclusion of the 4-1BB co-stimulatory domain or exclusion of a co-stimulatory domain, or blocking PD1 did not prevent CART cell depletion. Both CART cells and BsAb-T cells penetrated established subcutaneous human melanoma xenografts; while both induced tumor regression, BsAb was more efficient. The fate of T cells activated by BsAb differs substantially from that by CAR, translating into a more robust antitumor effect both in vitro and in vivo.

Effects of Zeaxanthin on Growth and Invasion of Human Uveal Melanoma in Nude Mouse Model.

Uveal melanoma cells were inoculated into the choroid of nude mice and treated with or without intraocular injection of zeaxanthin. After 21 days, mice were sacrificed and the eyes enucleated. Histopathological analysis was performed in hematoxylin and eosin stained frozen sections. Melanoma developed rapidly in the control group (without treatment of zeaxanthin). Tumor-bearing eye mass and tumor mass in the control group were significantly greater than those in zeaxanthin treated group. Melanoma in the controlled eyes occupied a large part of the eye, was epithelioid in morphology, and was with numerous mitotic figures. Scleral perforation and extraocular extension were observed in half of the eyes. Melanomas in zeaxanthin treated eyes were significantly smaller with many necrosis and apoptosis areas and no extraocular extension could be found. Quantitative image analysis revealed that the tumor size was reduced by 56% in eyes treated with low dosages of zeaxanthin and 92% in eyes treatment with high dosages of zeaxanthin, as compared to the controls. This study demonstrated that zeaxanthin significantly inhibits the growth and invasion of human uveal melanoma in nude mice, suggesting that zeaxanthin may be a promising agent to be explored for the prevention and treatment of uveal melanoma.

Rb suppresses human cone-precursor-derived retinoblastoma tumours.

Retinoblastoma is a childhood retinal tumour that initiates in response to biallelic RB1 inactivation and loss of functional retinoblastoma (Rb) protein. Although Rb has diverse tumour-suppressor functions and is inactivated in many cancers, germline RB1 mutations predispose to retinoblastoma far more strongly than to other malignancies. This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss, yet the nature of the circuitry and the cell type in which it operates have been unclear. Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion. Rb knockdown induced cone precursor proliferation in prospectively isolated populations and in intact retina. Proliferation followed the induction of E2F-regulated genes, and depended on factors having strong expression in maturing cone precursors and crucial roles in retinoblastoma cell proliferation, including MYCN and MDM2. Proliferation of Rb-depleted cones and retinoblastoma cells also depended on the Rb-related protein p107, SKP2, and a p27 downregulation associated with cone precursor maturation. Moreover, Rb-depleted cone precursors formed tumours in orthotopic xenografts with histological features and protein expression typical of human retinoblastoma. These findings provide a compelling molecular rationale for a cone precursor origin of retinoblastoma. More generally, they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

Death by water: precautionary water submersion for intravitreal injection of retinoblastoma eyes.

There is growing interest in intravitreal injections of chemotherapy for retinoblastoma. However, concerns for potential tumor seeding through the needle track has prompted the use of risk-reducing precautionary methods. Presented here is a novel technique, which can be easily replicated, requires minimal sophisticated equipment and with laboratory data supporting its concept. Sterile distilled water submersion for 3 minutes renders retinoblastoma cells nonviable and can be employed as a precautionary method following intravitreal injection in the technique described here.

Tumor-associated retinal astrocytes promote retinoblastoma cell proliferation through production of IGFBP-5.

Retinoblastomas consist of cone-like neoplastic cells and diverse non-neoplastic cells whose roles in tumorigenesis have not been defined. Here, we investigated the glial cells that constitute 2% to 3% of the cells in retinoblastoma tumors, including their origin, their relationship to a potential retinoblastoma stem cell population, and their effects on tumor cell proliferation. Retinoblastoma glia consistently expressed the retinal astrocyte marker Pax2 but inconsistently expressed the Müller cell and occasional astrocyte marker CRALBP. Many of the glia expressed the stem cell-associated Sox2 but nevertheless were non-neoplastic as they coexpressed Rb and/or retained two RB1 alleles. Conversely, the glia were distinct from the non-neoplastic cells that strongly expressed the stem cell-associated ABCG2. Adherent Pax2(+),Sox2(+),Rb(+) glia readily grew from explanted retinoblastomas and produced soluble factors that enhanced the proliferation of cocultured retinoblastoma cells. This effect was emulated by normal retinal glia and appeared to be mediated by insulin-like growth factor binding protein-5 (IGFBP-5), as it was mimicked by recombinant IGFBP-5 and mitigated by neutralizing IGFBP-5 antibody. As glia-derived IGFBP-5 was earlier found to promote photoreceptor survival, our findings indicate that retinal astrocytes enhance the proliferation of cone-like retinoblastoma cells by deploying a factor that also provides trophic support to the tumor cells' non-neoplastic counterparts. These observations suggest that a tissue-specific microenvironmental feature cooperates with oncogenic mutations in a cancer cell of origin to promote retinoblastoma tumorigenesis.

Retinoblastoma has properties of a cone precursor tumor and depends upon cone-specific MDM2 signaling.

Retinoblastomas result from the inactivation of the RB1 gene and the loss of Rb protein, yet the cell type in which Rb suppresses retinoblastoma and the circuitry that underlies the need for Rb are undefined. Here, we show that retinoblastoma cells express markers of postmitotic cone precursors but not markers of other retinal cell types. We also demonstrate that human cone precursors prominently express MDM2 and N-Myc, that retinoblastoma cells require both of these proteins for proliferation and survival, and that MDM2 is needed to suppress ARF-induced apoptosis in cultured retinoblastoma cells. Interestingly, retinoblastoma cell MDM2 expression was regulated by the cone-specific RXRgamma transcription factor and a human-specific RXRgamma consensus binding site, and proliferation required RXRgamma, as well as the cone-specific thyroid hormone receptor-beta2. These findings provide support for a cone precursor origin of retinoblastoma and suggest that human cone-specific signaling circuitry sensitizes to the oncogenic effects of RB1 mutations.