Natural Resistance to Methotrexate in Human Melanomas

Human melanomas are naturally resistant to methotrexate (MTX). The mechanism of intrinsic drug resistance has been explored in 3 melanoma cell lines not previously exposed to tins agent. All 3 lines exhibited relative MTX resistance with ID50 values of greater than 1 μm. Drug uptake studies were performed over an extracellular concentration range of 0.1 to 10 μm MTX. The uptake was linear over the initial 10min at all concentrations and subsequently reached plateau level only at the 10 μm Concentration. Lineweaver-Burke transformations yielded apparent Km (uptake) values of 1.4 to 5 μm, similar to data obtained from other human cell lines. The level of dihydrofolate reductase (DHFR) in the human melanoma cells ranged between 8.42 to 11.98 pmoles/mg protein. The melanoma DHFR levels are several fold higher than in MTX-sensitive human tumor lines and up to a hundred-fold higher than that measured in human brain tumor cells by our assay. The intrinsic resistance of these melanoma lines has therefore been attributed to elevated intracellular levels of DHFR

Induction of circulating phospholipase A2 by intravenous administration of recombinant human tumour necrosis factor

We have examined the effects of intravenous infusion of recombinant human tumour necrosis factor (rh-TNF) on serum activity of phospholipase A2 (PLA2) in patients with malignancies. Nine patients received a 24 h continuous intravenous infusion ranging from 1.0 × 105 U/m2 to 3.0 × 105 U/m2; 14 patients received a 5 day continuous intravenous infusion ranging from 0.5 × 105 U/m2/day to 3.0 105 U/m2/day. Twenty one of 23 patients responded with marked increases in serum PLA2 activity that were detectable 3 h after the beginning of the rh-TNF infusion and reached maximum levels at 18 h with a mean increase of 16.2-fold. In patients receiving a 5 day rh-TNF infusion, the highest levels of PLA2 were observed after the first day of infusion. Serum PLA2 activity declined continuously to 2.9-fold above baseline at the end of the infusion. A significant correlation was noted between the dose of infused rh-TNF and the maximum increase in PLA2 activity. To our knowledge, this is the first time that an association between intravenous TNF administration and induction of circulating PLA2 in man has been established

CBP501 induces immunogenic tumor cell death and CD8 T cell infiltration into tumors in combination with platinum, and increases the efficacy of immune checkpoint inhibitors against tumors in mice

CBP501, a calmodulin-binding peptide, is an anti-cancer drug candidate and functions as an enhancer of platinum uptake into cancer cells. Here we show that CBP501 promotes immunogenic cell death (ICD) in combination with platinum agents. CBP501 enhanced a clinically relevant low dose of cisplatin (CDDP) in vitro as evidenced by upregulation of ICD markers, including cell surface calreticulin exposure and release of high-mobility group protein box-1. Synergistic induction of ICD by CDDP plus CBP501 as compared to CDDP alone was confirmed in the well-established vaccination assay. Furthermore, cotreatment of CDDP plus CBP501 significantly reduced the tumor growth and upregulated the percentage of tumor infiltrating CD8+ T cell in vivo. Importantly, the antitumor effect of CDDP plus CBP501 was significantly reduced by anti-CD8 antibody treatment. Based on this novel effect of CBP501, we analyzed the combination treatment with immune checkpoint inhibitors in vivo. Mice treated with CBP501 in combination with CDDP and anti-PD-1 or anti-PD-L1 showed an additive antitumor effect. These results support the conclusion that CBP501 enhances CDDP-induced ICD in vitro and in vivo. The findings also support the further clinical development of the CBP501 for enhancing the antitumor activity of immune checkpoint inhibitors in combination with CDDP

CBP501 inhibits EGF-dependent cell migration, invasion and epithelial-to–mesenchymal transition of non-small cell lung cancer cells by blocking KRas to calmodulin binding

The anti-cancer agent CBP501 binds to calmodulin (CaM). Recent studies showed that migration and metastasis are inhibited by several CaM antagonists. However, there is no available evidence that CBP501 has similar effects. Here we found that CBP501 inhibits migration of non-small cell lung cancer (NSCLC) cells in vitro, even in the presence of migration inducing factors such as WNT, IL-6, and several growth factors. CBP501 also inhibited epidermal growth factor (EGF) enhanced invasion and the epithelial-to-mesenchymal transition (EMT), and this inhibition was accompanied by (i) suppression of Akt and ERK1/2 phosphorylation, and (ii) suppression of expression of transcription factor Zeb1 and the mesenchymal marker Vimentin. A pull down analysis performed using sepharose-immobilized CaM showed that CBP501 blocks the interaction between CaM and KRas. Furthermore, EGF induced Akt activation and cell migration was effectively suppressed by KRas down-regulation in NSCLC cells. Stable knockdown of KRas also made cells insensitive to CBP501’s inhibition of growth factor-induced migration. Taken together, these results indicate that CBP501 inhibits binding of CaM with KRas and thereby suppresses the PI3K/AKT pathway, migration, invasion and EMT. These findings have identified a previously unrecognized effect of CBP501 on downstream KRas signaling mechanisms involving EMT and invasion, and provide support for the further clinical development of this agent

MUC1-C Oncoprotein Regulates Glycolysis and Pyruvate Kinase m2 Activity in Cancer Cells

Aerobic glycolysis in cancer cells is regulated by multiple effectors that include Akt and pyruvate kinase M2 (PKM2). Mucin 1 (MUC1) is a heterodimeric glycoprotein that is aberrantly overexpressed by human breast and other carcinomas. Here we show that transformation of rat fibroblasts by the oncogenic MUC1-C subunit is associated with Akt-mediated increases in glucose uptake and lactate production, consistent with the stimulation of glycolysis. The results also demonstrate that the MUC1-C cytoplasmic domain binds directly to PKM2 at the B- and C-domains. Interaction between the MUC1-C cytoplasmic domain Cys-3 and the PKM2 C-domain Cys-474 was found to stimulate PKM2 activity. Conversely, epidermal growth factor receptor (EGFR)-mediated phosphorylation of the MUC1-C cytoplasmic domain on Tyr-46 conferred binding to PKM2 Lys-433 and inhibited PKM2 activity. In human breast cancer cells, silencing MUC1-C was associated with decreases in glucose uptake and lactate production, confirming involvement of MUC1-C in the regulation of glycolysis. In addition, EGFR-mediated phosphorylation of MUC1-C in breast cancer cells was associated with decreases in PKM2 activity. These findings indicate that the MUC1-C subunit regulates glycolysis and that this response is conferred in part by PKM2. Thus, the overexpression of MUC1-C oncoprotein in diverse human carcinomas could be of importance to the Warburg effect of aerobic glycolysis

MUC1-associated proliferation signature predicts outcomes in lung adenocarcinoma patients

Background: MUC1 protein is highly expressed in lung cancer. The cytoplasmic domain of MUC1 (MUC1-CD) induces tumorigenesis and resistance to DNA-damaging agents. We characterized MUC1-CD-induced transcriptional changes and examined their significance in lung cancer patients. Methods: Using DNA microarrays, we identified 254 genes that were differentially expressed in cell lines transformed by MUC1-CD compared to control cell lines. We then examined expression of these genes in 441 lung adenocarcinomas from a publicly available database. We employed statistical analyses independent of clinical outcomes, including hierarchical clustering, Student's t-tests and receiver operating characteristic (ROC) analysis, to select a seven-gene MUC1-associated proliferation signature (MAPS). We demonstrated the prognostic value of MAPS in this database using Kaplan-Meier survival analysis, log-rank tests and Cox models. The MAPS was further validated for prognostic significance in 84 lung adenocarcinoma patients from an independent database. Results: MAPS genes were found to be associated with proliferation and cell cycle regulation and included CCNB1, CDC2, CDC20, CDKN3, MAD2L1, PRC1 and RRM2. MAPS expressors (MAPS+) had inferior survival compared to non-expressors (MAPS-). In the initial data set, 5-year survival was 65% (MAPS-) vs. 45% (MAPS+, p < 0.0001). Similarly, in the validation data set, 5-year survival was 57% (MAPS-) vs. 28% (MAPS+, p = 0.005). Conclusions: The MAPS signature, comprised of MUC1-CD-dependent genes involved in the control of cell cycle and proliferation, is associated with poor outcomes in patients with adenocarcinoma of the lung. These data provide potential new prognostic biomarkers and treatment targets for lung adenocarcinoma

Emergence of MUC1 in Mammals for Adaptation of Barrier Epithelia

The mucin 1 (MUC1) gene was discovered based on its overexpression in human breast cancers. Subsequent work demonstrated that MUC1 is aberrantly expressed in cancers originating from other diverse organs, including skin and immune cells. These findings supported a role for MUC1 in the adaptation of barrier tissues to infection and environmental stress. Of fundamental importance for this evolutionary adaptation was inclusion of a SEA domain, which catalyzes autoproteolysis of the MUC1 protein and formation of a non-covalent heterodimeric complex. The resulting MUC1 heterodimer is poised at the apical cell membrane to respond to loss of homeostasis. Disruption of the complex releases the MUC1 N-terminal (MUC1-N) subunit into a protective mucous gel. Conversely, the transmembrane C-terminal (MUC1-C) subunit activates a program of lineage plasticity, epigenetic reprogramming and repair. This MUC1-C-activated program apparently evolved for barrier tissues to mount self-regulating proliferative, inflammatory and remodeling responses associated with wound healing. Emerging evidence indicates that MUC1-C underpins inflammatory adaptation of tissue stem cells and immune cells in the barrier niche. This review focuses on how prolonged activation of MUC1-C by chronic inflammation in these niches promotes the cancer stem cell (CSC) state by establishing auto-inductive nodes that drive self-renewal and tumorigenicity

Chronic activation of MUC1-C in wound repair promotes progression to cancer stem cells

The mucin 1 (MUC1) gene emerged in mammals to afford protection of barrier epithelial tissues from the external environment. MUC1 encodes a transmembrane C-terminal (MUC1-C) subunit that is activated by loss of homeostasis and induces inflammatory, proliferative, and remodeling pathways associated with wound repair. As a consequence, chronic activation of MUC1-C promotes lineage plasticity, epigenetic reprogramming, and carcinogenesis. In driving cancer progression, MUC1-C is imported into the nucleus, where it induces NF-κB inflammatory signaling and the epithelial-mesenchymal transition (EMT). MUC1-C represses gene expression by activating (i) DNA methyltransferase 1 (DNMT1) and DNMT3b, (ii) Polycomb Repressive Complex 1 (PRC1) and PRC2, and (iii) the nucleosome remodeling and deacetylase (NuRD) complex. PRC1/2-mediated gene repression is counteracted by the SWI/SNF chromatin remodeling complexes. MUC1-C activates the SWI/SNF BAF and PBAF complexes in cancer stem cell (CSC) models with the induction of genome-wide differentially accessible regions and expressed genes. MUC1-C regulates chromatin accessibility of enhancer-like signatures in association with the induction of the Yamanaka pluripotency factors and recruitment of JUN and BAF, which promote increases in histone activation marks and opening of chromatin. These and other findings described in this review have uncovered a pivotal role for MUC1-C in integrating lineage plasticity and epigenetic reprogramming, which are transient in wound repair and sustained in promoting CSC progression