Analysis of the inhibitory effect of catalytic activity of aldehyde dehydrogenase (ALDH) on the proliferation and viability of melanoma cells

Dehydrogenaza aldehydowa (ALDH) odgrywa rolę w chemooporności nowotworów – inhibicja jej aktywności może prowadzić do spadku żywotności i tempa proliferacji komórek w wielu typach nowotworów, natomiast wysoka aktywność tego enzymu jest uznawana za marker nowotworowych komórek macierzystych, m. in. komórek czerniaka. Zaobserwowano także, że w przypadku tego nowotworu nadekspresja oksygenazy hemowej-1 (HO-1), enzymu o właściwościach cytoprotekcyjnych, może zwiększać jego agresywność. Celem przeprowadzonych w tej pracy doświadczeń była analiza wpływu inhibicji ALDH na komórki czerniaka. Wykonano w tym celu testy żywotności i proliferacji oraz aktywności metabolicznej. Ze względu na to, że w dotychczasowych badaniach wykazano, że populacja komórek czerniaka charakteryzująca się wysoką aktywnością ALDH ma podwyższoną ekspresję HO-1 sprawdzono także wpływ zahamowania aktywności tego enzymu na ekspresję i aktywność HO-1. Na podstawie wykonanych doświadczeń stwierdzono, że inhibicja ALDH w komórkach czerniaka nie wpływa ekspresję i aktywność HO-1, ani na żywotność i proliferację, co może wskazywać na to, że zahamowanie aktywności ALDH nie stanowiłoby dobrego potencjalnego obiektu w terapiach przeciwnowotworowych czerniaka.Aldehyde dehydrogenase (ALDH) plays a role in the chemoresistance of cancer - its inhibition can lead to a decrease of viability and proliferation rate of cells in multiple types of tumors. The high activity of this enzyme is also considered to be a marker of cancer stem cells, i.a. melanoma cells. It was observed that in melanoma overexpression of heme oxygenase-1 (HO-1), enzyme which has cytoprotective properties, may increase its aggressiveness.The aim of this work was to analyze the effect of the inhibition of ALDH on melanoma cells. In order to determine this effect viability, proliferation and metabolic activity were tested. Previous studies have shown that melanoma cells with high activity of ALDH have increased HO-1 expression, thus the influence of inhibition of this enzyme on expression and activity of HO-1 was examined.In conclusion, it was found that the inhibition of ALDH in melanoma cells affects neither the expression and activity of HO-1, nor cell viability and proliferation, which may indicate that inhibition of ALDH would not be a potential target of anti-melanoma therapies

Effect of heme oxygenase-1 expression level on pigmentation of murine melanoma cells

Aktywność enzymów antyoksydacyjnych pełni ważną rolę w rozwoju nowotworów. Jednym z takich enzymów jest oksygenaza hemowa-1 (HO-1), odpowiedzialna za rozkład hemu do tlenku węgla, jonów żelaza (Fe2+) i biliwerdyny. Wcześniejsze prace pokazały, że nadekspresja HO-1 w komórkach czerniaka działa cytoprotekcyjnie i zwiększa agresywność nowotworu, w tym oporność na stres oksydacyjny i zdolność do przerzutowania. Zmniejsza jednocześnie wrażliwość komórek na różne formy terapii. Zmniejszenie wrażliwości obserwowano również przy nasilonej syntezie melaniny, natomiast hamowaniu melanogenezy towarzyszył wzrost wrażliwości. Ponieważ są publikacje sugerujące wpływ HO-1 na fizjologiczną melanogenezę, celem prezentowanej pracy było sprawdzenie, na ile HO-1 może wpływać na melanogenezę w komórkach czerniaka. Badania wykonano na stabilnych liniach komórkowych mysiego czerniaka B16(F10) o różnych poziomach HO-1: obniżonym (po transdukcji retrowirusowej shRNA-HO1, linia shHO-1), endogennym (po transdukcji retrowirusowej shRNA-scrambled, linia scrambled oraz linia WT) oraz podwyższonym (po transdukcji retrowirusowej cDNA-HO-1, linia HO-1). Pierwszą obserwacją były różnice w pigmentacji komórek, przy czym poziom pigmentacji korelował z ekspresją HO-1. Najniższy był w linii shHO-1, a najwyższy w komórkach z nadekspresją HO-1. Mimo różnic w pigmentacji, nie zaobserwowano między liniami różnic w ekspresji genów melanogenezy, zarówno na poziomie mRNA, jak i białka. Badane linie różniły się natomiast aktywnością tyrozynazy, która również korelowała z poziomem HO-1 w komórkach. Stymulacja komórek B16(F10) do produkcji melaniny pokazała, że linia shHO 1 ma mniejszą zdolność do intensywnej pigmentacji w porównaniu z komórkami WT i HO-1. Pomiar zawartości melaniny w pożywce wykazał, że komórki shHO-1 silniej wydzielają melaninę, a efekt ten jest odwracany przez witaminę B3, inhibitor transportu melanosomów. Podsumowując, aktywność HO-1 w komórkach mysiego czerniaka zwiększa pigmentację komórek poprzez nasilenie produkcji melaniny i podniesienie aktywności tyrozynazy.The activity of antioxidant enzymes plays an important role in the development of cancer. One of such enzymes is heme-1 oxygenase (HO-1), which is responsible for the degradation of heme to carbon monoxide, divalent iron ions (Fe2+) and biliverdin. Earlier studies have shown that overexpression of HO-1 in melanoma cells is cytoprotective and increases tumor aggressiveness, including resistance to oxidative stress and metastasis. At the same time, it reduces the sensitivity of cells to various forms of therapy. A decrease in sensitivity was also observed with increased melanin synthesis, while inhibition of melanogenesis was accompanied by an increase in sensitivity. Because there are studies suggesting the impact of HO-1 on physiological melanogenesis, the aim of this study was to see how much HO-1 can affect melanogenesis in melanoma cells.The tests were performed on stable B16(F10) murine melanoma cell lines with different HO-1 levels: reduced (after shRNA-HO1 retroviral transduction; shHO-1 cell line), endogenous (after shRNA-scrambled retroviral transduction; scrambled cell line and WT cell line) and elevated (after retroviral transduction of cDNA-HO-1; HO-1 cell line). The first observation was differences in cell pigmentation, with the level of pigmentation correlating with HO-1 expression. The lowest was in the shHO-1 cells, and the highest in HO-1 overexpressing cells. Despite differences in pigmentation, no differences in melanogenesis gene expression lines were observed at both mRNA and protein levels. However, the tested lines differed in tyrosinase activity, which also correlated with the level of HO-1 in the cells. Induction of melanin production in B16(F10) cells showed that the shHO-1 cells have decreased pigmentation capacity compared to WT and HO-1 cells. Measurement of melanin content in the medium showed that shHO-1 cells secrete melanin more strongly, and this effect is reversed by vitamin B3, an inhibitor of melanosome transport.In summary, HO-1 activity in mouse melanoma cells increases cell pigmentation by enhancing melanin production and increasing tyrosinase activity

Heme oxygenase-1 has a greater effect on melanoma stem cell properties than the expression of melanoma-initiating cell markers

Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDHhigh) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation

Slow-cycling murine melanoma cells display plasticity and enhanced tumorigenicity in syngeneic transplantation assay

Slow-cycling cancer cells (SCC) contribute to the aggressiveness of many cancers, and their invasiveness and chemoresistance pose a great therapeutic challenge. However, in melanoma, their tumor-initiating abilities are not fully understood. In this study, we used the syngeneic transplantation assay to investigate the tumor-initiating properties of melanoma SCC in the physiologically relevant in vivo settings. For this we used B16-F10 murine melanoma cell line where we identified a small fraction of SCC. We found that, unlike human melanoma, the murine melanoma SCC were not marked by the high expression of the epigenetic enzyme Jarid1b. At the same time, their slow-cycling phenotype was a temporary state, similar to what was described in human melanoma. Progeny of SCC had slightly increased doxorubicin resistance and altered expression of melanogenesis genes, independent of the expression of cancer stem cell markers. Single-cell expansion of SCC revealed delayed growth and reduced clone formation when compared to non-SCC, which was further confirmed by an in vitro limiting dilution assay. Finally, syngeneic transplantation of 10 cells in vivo established that SCC were able to initiate growth in primary recipients and continue growth in the serial transplantation assay, suggesting their self-renewal nature. Together, our study highlights the high plasticity and tumorigenicity of murine melanoma SCC and suggests their role in melanoma aggressiveness

Proximity ligation assay detection of protein-DNA interactions : is there a link between heme oxygenase-1 and G-quadruplexes?

G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1−/− mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4–protein colocalizations can be quantitatively analyzed using PLA, even in rare cells

Histone H3K27 demethylase KDM6A is an epigenetic gatekeeper of mTORC1 signalling in cancer

ObjectiveLarge-scale genome sequencing efforts of human tumours identified epigenetic modifiers as one of the most frequently mutated gene class in human cancer. However, how these mutations drive tumour development and tumour progression are largely unknown. Here, we investigated the function of the histone demethylase KDM6A in gastrointestinal cancers, such as liver cancer and pancreatic cancer.DesignGenetic alterations as well as expression analyses of KDM6A were performed in patients with liver cancer. Genetic mouse models of liver and pancreatic cancer coupled with Kdm6a-deficiency were investigated, transcriptomic and epigenetic profiling was performed, and in vivo and in vitro drug treatments were conducted.ResultsKDM6A expression was lost in 30% of patients with liver cancer. Kdm6a deletion significantly accelerated tumour development in murine liver and pancreatic cancer models. Kdm6a-deficient tumours showed hyperactivation of mTORC1 signalling, whereas endogenous Kdm6a re-expression by inducible RNA-interference in established Kdm6a-deficient tumours diminished mTORC1 activity resulting in attenuated tumour progression. Genome-wide transcriptional and epigenetic profiling revealed direct binding of Kdm6a to crucial negative regulators of mTORC1, such as Deptor, and subsequent transcriptional activation by epigenetic remodelling. Moreover, in vitro and in vivo genetic epistasis experiments illustrated a crucial function of Deptor and mTORC1 in Kdm6a-dependent tumour suppression. Importantly, KDM6A expression in human tumours correlates with mTORC1 activity and KDM6A-deficient tumours exhibit increased sensitivity to mTORC1 inhibition.ConclusionKDM6A is an important tumour suppressor in gastrointestinal cancers and acts as an epigenetic toggle for mTORC1 signalling. Patients with KDM6A-deficient tumours could benefit of targeted therapy focusing on mTORC1 inhibition

Etiology-independent activation of the LTβ-LTβR-RELB axis drives aggressiveness and predicts poor prognosis in Hepatocellular carcinoma

BACKGROUND AIMS: Hepatocellular carcinoma (HCC) is the most common primary liver tumor with an increasing incidence worldwide. HCC is a heterogeneous malignancy and develops usually in a chronically injured liver. The nuclear factor kappa B (NF-κB) signaling network consists of a canonical and a non-canonical branch. An activation of canonical NF-κB in HCC is documented. However, a functional and clinically relevant role of non-canonical NF-κB and its downstream effectors is not established. APPROACH RESULTS: Four human HCC cohorts (total n=1,462) and four mouse HCC models were assessed for expression and localization of NF-κB signaling components and activating ligands. In vitro, NF-κB signaling, proliferation and cell death were measured, proving a pro-proliferative role of RELB activated via NIK. In vivo, Lymphotoxin beta (LTβ) was identified as predominant inducer of RELB activation. Importantly, hepatocyte-specific RELB knockout in a murine HCC model led to a lower incidence compared to controls and lower maximal tumor diameters. In silico, RELB activity and RELB directed transcriptomics were validated on the TCGA HCC cohort using inferred protein activity and Gene Set Enrichment Analysis (GSEA). In RELB-active HCC, pathways mediating proliferation were significantly activated. In contrast to RELA, nuclear enrichment of non-canonical RELB expression identified patients with a poor prognosis in an etiology-independent manner. Moreover, RELB activation was associated with malignant features metastasis and recurrence. CONCLUSIONS: This study demonstrates a prognostically relevant, etiology-independent and cross-species consistent activation of a LTβ/LTβR/RELB axis in hepatocarcinogenesis. These observations may harbor broad implications for HCC, including possible clinical exploitation