Cellular Tumor Models Research Articles

Inducible overexpression of a FAM3C/ILEI transgene has pleiotropic effects with shortened life span, liver fibrosis and anemia in mice.

FAM3C/ILEI is an important factor in epithelial-to-mesenchymal transition (EMT) induction, tumor progression and metastasis. Overexpressed in many cancers, elevated ILEI levels and secretion correlate with poor patient survival. Although ILEI's causative role in invasive tumor growth and metastasis has been demonstrated in several cellular tumor models, there are no available transgenic mice to study these effects in the context of a complex organism. Here, we describe the generation and initial characterization of a Tet-ON inducible Fam3c/ILEI transgenic mouse strain. We find that ubiquitous induction of ILEI overexpression (R26-ILEIind) at weaning age leads to a shortened lifespan, reduced body weight and microcytic hypochromic anemia. The anemia was reversible at a young age within a week upon withdrawal of ILEI induction. Vav1-driven overexpression of the ILEIind transgene in all hematopoietic cells (Vav-ILEIind) did not render mice anemic or lower overall fitness, demonstrating that no intrinsic mechanisms of erythroid development were dysregulated by ILEI and that hematopoietic ILEI hyperfunction did not contribute to death. Reduced serum iron levels of R26-ILEIind mice were indicative for a malfunction in iron uptake or homeostasis. Accordingly, the liver, the main organ of iron metabolism, was severely affected in moribund ILEI overexpressing mice: increased alanine transaminase and aspartate aminotransferase levels indicated liver dysfunction, the liver was reduced in size, showed increased apoptosis, reduced cellular iron content, and had a fibrotic phenotype. These data indicate that high ILEI expression in the liver might reduce hepatoprotection and induce liver fibrosis, which leads to liver dysfunction, disturbed iron metabolism and eventually to death. Overall, we show here that the novel Tet-ON inducible Fam3c/ILEI transgenic mouse strain allows tissue specific timely controlled overexpression of ILEI and thus, will serve as a versatile tool to model the effect of elevated ILEI expression in diverse tissue entities and disease conditions, including cancer.

Abstract LB-110: Comparison of the effects of tubulin-targeting and DNA-damaging agents on the modulation and distribution of HSP90, pHSP90αT5/7, and tubulin in cancer cells

Abstract Microtubules (MTs) are essential architectural components of cells whose dynamic structures are critical for multiple functions including cell division, intracellular signaling, and trafficking. Many proteins are associated with MTs or their motors, including molecular chaperones such as heat shock protein 90 (HSP90), a key component of the cellular proteostatic machinery. The coordinated interplay of these processes is crucial for maintaining normal or malignant cell phenotypes. Alterations in protein levels, their localization and/or post-translational modifications (PTMs), such as phosphorylation or acetylation, are recognized as targets for disruption and may impact cell viability. To that end, we investigated the levels of HSP90 and pHSP90αT5/7 (a unique PTM previously associated with DNA damage) in both prostate (22RV1, PC3), and colorectal (HCT116wt, HCT116 DNA-PK -/-) carcinoma cell lines after treatment with microtubule-targeting agents (MTAs) or DNA-damaging agents (DDAs). We observed a dose dependent increase in the level of pHSP90αT5/7 protein in each of the cell lines treated with an MTA (paclitaxel (PTX), vincristine) or a DDA (gemcitabine, oxaliplatin) compared to the untreated control, whereas the levels of HSP90 remained monotonous throughout. The increases in pHSP90αT5/7 were independent of DNA-PK status for the HCT116 cells. These observations were accompanied by increasing levels of γ-H2AX and acetylated tubulin, the latter prominent following treatment with PTX. When cell lysates were separated into cytoplasmic (C) and nuclear (N) fractions, HSP90 and pHSP90αT5/7 were located predominantly in the C fractions while acetylated tubulin was surprisingly distributed between the C and N fractions as was γ-H2AX. Markers of cytoplasmic localization, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the voltage-dependent anion channel (VDAC), and of nuclear localization, including phospho-Histone H3 and acetylated-Histone H3, were each segregated to their appropriate cellular compartments. HSP90α was previously shown to be a substrate and chaperone of the nuclear kinase DNA-PK and its DNA-PK dependent phosphorylation in the nucleus at T5 and T7 was necessary for the apoptotic response to TRAIL (Solier S, et al PNAS, 105:32, 2012). In contrast, our data demonstrate that HSP90αT5/7 phosphorylation also occurs in response to MTA, but is localized in cytosol and is independent of DNA-PK. Meanwhile, others have reported that tubulin acetylation favors HSP90 recruitment to MTs, stimulating the signaling function of HSP90 clients (Giustiniani, J, et al Cell Sig, 21, 2009). The unique effects of MTAs and DDAs on pHSP90αT5/7 and tubulin acetylation have implications for malignant cell survival and drug resistance mechanisms. Experiments are underway to determine the significance of cytosolic pHsp90αT5/7 and nuclear acetylated tubulin in these cellular tumor models. Citation Format: Marianne S. Poruchynsky, Michael Moses, Len Neckers. Comparison of the effects of tubulin-targeting and DNA-damaging agents on the modulation and distribution of HSP90, pHSP90αT5/7, and tubulin in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-110. doi:10.1158/1538-7445.AM2017-LB-110

The small conductance calcium-activated potassium channel 3 (SK3) is a molecular target for Edelfosine to reduce the invasive potential of urothelial carcinoma cells.

Metastasis is the survival-determining factor in urothelial carcinoma (UC) of the urinary bladder. The small conductance calcium-activated potassium channel 3 (SK3) enhances tumor cell invasion in breast cancer and malignant melanoma. Since Edelfosine, a glycerophospholipid with antitumoral properties, effectively inhibits SK3 channel activity, our goal was to evaluate SK3 as a potential molecular target to inhibit the gain of an invasive phenotype in UC. SK3 protein expression was analyzed in 208 tissue samples and UC cell lines. Effects of Edelfosine on SK3 expression and intracellular calcium levels as well as on cell morphology, cell survival and proliferation were assessed using immunoblotting, potentiometric fluorescence microscopy, and clonogenic/cell survival assay; furthermore, we analyzed the effect of Edelfosine and SK3 RNAi knockdown on tumor cell migration and invasion in vitro and in vivo. We found that SK3 is strongly expressed in muscle-invasive UC and in the RT112 cellular tumor model. Higher concentrations of Edelfosine have a strong antitumoral effect on UC cells, while 1μM effectively inhibits migration/invasion of UC cells in vitro and in vivo comparable to the SK3 knockdown phenotype. Taken together, our results show strong expression of SK3 in muscle-invasive UC, consistent with the postulated role of the protein in tumor cell invasion. Edelfosine is able to effectively inhibit migration and invasion of UC cells in vitro and in vivo in an SK3-dependent way, pointing towards a possible role for Edelfosine as an antiinvasive drug to effectively inhibit UC cell invasion and metastasis.

Radiosensitization and downregulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) upon inhibition of mitogen/extracellular signal-regulated kinase (MEK) in malignant melanoma cells.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an important cofactor in the p53-mediated DNA damage response pathway upon ionizing radiation (IR) and exerts anti-apoptotic effects also independent of p53 pathway activation. Furthermore, hnRNP K is overexpressed in various neoplasms including malignant melanoma (MM). Here, we investigate the role of hnRNP K in the radioresistance of MM cells. Our results show cytoplasmic expression of hnRNP K in human MM surgical specimens, but not in benign nevi, and a quick dose- and time-dependent upregulation in response to IR accompanied by cytoplasmic redistribution of the protein in the IPC-298 cellular tumor model carrying an activating NRAS mutation (p.Q61L). SiRNA-based knockdown of hnRNP K induced a delayed decline in γH2AX/53BP1-positive DNA repair foci upon IR. Pharmacological interference with MAPK signaling abrogated ERK phosphorylation, diminished cellular hnRNP K levels, impaired γH2AX/53BP1-foci repair and proliferative capability and increased apoptosis comparable to the observed hnRNP K knockdown phenotype in IPC-298 cells. Our results indicate that pharmacological interference with MAPK signaling increases vulnerability of NRAS-mutant malignant melanoma cells to ionizing radiation along with downregulation of endogenous hnRNP K and point towards a possible use for combined MEK inhibition and localized radiation therapy of MM in the NRAS-mutant setting where BRAF inhibitors offer no clinical benefit.

Metastatic phenotype of murine tumor cells expressing different cooperating oncogenes.

Four murine cellular tumor models expressing various combinations of oncogenes (SV40 large T and v-Ha-ras, SV40 large T and v-src, SV40 large T and neu, adenovirus EIA and v-Ha-ras) induce sarcoma when they are inoculated s.c. into the DBA/2 syngenic mice. The metastatic patterns, distribution and fate of these tumor cells transplanted by two different routes into syngenic DBA/2 mice have been studied. All the tumor cell lines except EIA-ras, induce massive overt artificial metastases principally in the lung after i.v. injection. In s.c. tumor-bearing mice, a few resting cells colonize the lung as micrometastases. When removed from this tissue context and injected s.c. these cells regain their proliferative potential and grow as local tumors which again give rise to occult pulmonary micrometastases.