MLK4 regulates DNA damage response and promotes triple-negative breast cancer chemoresistance.

Chemoresistance constitutes a major challenge in the treatment of triple-negative breast cancer (TNBC). Mixed-Lineage Kinase 4 (MLK4) is frequently amplified or overexpressed in TNBC where it facilitates the aggressive growth and migratory potential of breast cancer cells. However, the functional role of MLK4 in resistance to chemotherapy has not been investigated so far. Here, we demonstrate that MLK4 promotes TNBC chemoresistance by regulating the pro-survival response to DNA-damaging therapies. We observed that MLK4 knock-down or inhibition sensitized TNBC cell lines to chemotherapeutic agents in vitro. Similarly, MLK4-deficient cells displayed enhanced sensitivity towards doxorubicin treatment in vivo. MLK4 silencing induced persistent DNA damage accumulation and apoptosis in TNBC cells upon treatment with chemotherapeutics. Using phosphoproteomic profiling and reporter assays, we demonstrated that loss of MLK4 reduced phosphorylation of key DNA damage response factors, including ATM and CHK2, and compromised DNA repair via non-homologous end-joining pathway. Moreover, our mRNA-seq analysis revealed that MLK4 is required for DNA damage-induced expression of several NF-кB-associated cytokines, which facilitate TNBC cells survival. Lastly, we found that high MLK4 expression is associated with worse overall survival of TNBC patients receiving anthracycline-based neoadjuvant chemotherapy. Collectively, these results identify a novel function of MLK4 in the regulation of DNA damage response signaling and indicate that inhibition of this kinase could be an effective strategy to overcome TNBC chemoresistance.

Inhibition of the ʟ-glutamine transporter ASCT2 sensitizes plasma cell myeloma cells to proteasome inhibitors.

Proteasome inhibitors (PIs), used in the treatment of plasma cell myeloma (PCM), interfere with the degradation of misfolded proteins leading to activation of unfolded protein response (UPR) and cell death. However, despite initial strong antimyeloma effects, PCM cells eventually develop acquired resistance to PIs. The pleiotropic role of ʟ-glutamine (Gln) in cellular functions makes inhibition of Gln metabolism a potentially good candidate for combination therapy. Here, we show that PCM cells, both sensitive and resistant to PIs, express membrane Gln transporter (ASCT2), require extracellular Gln for survival, and are sensitive to ASCT2 inhibitors (ASCT2i). ASCT2i synergistically potentiate the cytotoxic activity of PIs by inducing apoptosis and modulating autophagy. Combination of ASCT2 inhibitor V9302 and proteasome inhibitor carfilzomib upregulates the intracellular levels of ROS and oxidative stress markers and triggers catastrophic UPR as shown by upregulated spliced Xbp1 mRNA, ATF3 and CHOP levels. Moreover, analysis of RNA sequencing revealed that the PI in combination with ASCT2i reduced the levels of Gln metabolism regulators such as MYC and NRAS. Analysis of PCM patients' data revealed that upregulated ASCT2 and other Gln metabolism regulators are associated with advanced disease stage and with PIs resistance. Altogether, we identified a potent therapeutic approach that may prevent acquired resistance to PIs and may contribute to the improvement of treatment of patients suffering from PCM.

ALK-rearranged renal cell carcinomas in Polish population.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, the activation of which is considered an important event in the pathogenesis of several neoplasms and a predictive factor for the targeted therapy with ALK inhibitors. Thus far, ALK rearrangements have been identified in 22 renal cell carcinomas in both pediatric and adult patients. We evaluated the incidence of ALK rearrangement-associated RCC in adult Central European population. An immunohistochemical evaluation of 1019 kidney tumors was performed with use of three different clones of anti-ALK antibodies. None of the tested samples showed positive staining, which suggests that the incidence of ALK rearrangement-associated renal cell carcinomas is significantly lower in the Polish population, and indicates a potential association between ethnicity and occurrence of these rare neoplasms.

Upregulation of MLK4 promotes migratory and invasive potential of breast cancer cells.

Metastasis to distant organs is a major cause for solid cancer mortality, and the acquisition of migratory and invasive phenotype is a key factor in initiation of malignancy. In this study we investigated the contribution of Mixed-Lineage Kinase 4 (MLK4) to aggressive phenotype of breast cancer cells. Our TCGA cancer genomic data analysis revealed that amplification or mRNA upregulation of MLK4 occurred in 23% of invasive breast carcinoma cases. To find the association between MLK4 expression and the specific subtype of breast cancer, we performed a transcriptomic analysis of multiple datasets, which showed that MLK4 is highly expressed in triple-negative breast cancer compared to other molecular subtypes. Depletion of MLK4 in cell lines with high MLK4 expression impaired proliferation and anchorage-dependent colony formation. Moreover, silencing of MLK4 expression significantly reduced the migratory potential and invasiveness of breast cancer cells as well as the number of spheroids formed in 3D cultures. These in vitro findings translate into slower rate of tumor growth in mice upon MLK4 knock-down. Furthermore, we established that MLK4 activates NF-κB signaling and promotes a mesenchymal phenotype in breast cancer cells. Immunohistochemical staining of samples obtained from breast cancer patients revealed a strong positive correlation between high expression of MLK4 and metastatic potential of tumors, which was predominantly observed in TNBC subgroup. Taken together, our results show that high expression of MLK4 promotes migratory and invasive phenotype of breast cancer and may represent a novel target for anticancer treatment.

4-(Methylthio)butyl isothiocyanate inhibits the proliferation of breast cancer cells with different receptor status.

BACKGROUND: Epidemiological studies indicate that the consumption of Brassicaceae plants, a rich source of biologically active isothiocyanates (ITCs), may effectively reduce cancer risk. In the current study, we evaluated the anticancer potential of 4-(methylthio)butyl ITC (erucin, ERN) against three phenotypically different breast cancer cell lines: MDA-MB-231, SKBR-3 and T47D. METHODS: The effect of ERN on the viability of breast cancer cells was evaluated using sulforhodamine B and clonogenic assays, and acridine orange/ethidium bromide staining. Cell cycle was investigated using flow cytometry. The status of signaling molecules was examined by western blot analysis. RESULTS: ERN decreased the viability of all tested cancer cell lines in a concentration-dependent manner; this effect was much weaker in normal breast cells (MCF-10A). ERN induced cell cycle arrest in the G2/M phase, down-regulated the phosphorylation of S6 ribosomal protein in all tested breast cancer cell lines, and reduced HER2 receptor levels in SKBR-3 cells. A 24-h treatment with lower concentrations of ERN (5-20µM) induced apoptosis; higher ERN concentrations (40µM) induced necrosis. The latter also irreversibly inhibited the proliferative potential of cancer cells. CONCLUSION: ERN effectively inhibits proliferation of breast cancer cells irrespectively of their receptor status.

ALK-positive cancer: still a growing entity.

 Since the discovery of ALK-positive anaplastic large-cell lymphoma in 1994 many other types of tumors showing ALK expression were disclosed. They form a heterogeneous group, including lung, renal and soft tissue tumors. The biological function of ALK, its role in carcinogenesis and impact exerted on the clinical outcome have been studied by many research groups. New drugs specifically dedicated for ALK inhibition, for example, crizotinib, have been synthesized and have become a viable treatment option for ALK-positive lung adenocarcinoma, and potentially for other ALK-positive cancers. This review summarizes the current state of knowledge concerning ALK-positive neoplasms, focusing on the clinical aspects of the subject.