The dysregulation of transforming growth factor-β (TGF-β) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-β treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation. We first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038). ABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.

Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFβ signaling and induced JAK1–STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1–STAT3 was associated with increased levels of AXL in treatment-naïve tumors. Tumors with high AXL, TGFβ, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelial-to-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-naïve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFβ, and JAK1–STAT3 signal activation in select tumors that may be targeted by combined AXL–JAK1 inhibition. Significance: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.

The humanized monoclonal antibody-drug conjugate trastuzumab emtansine (T-DM1, Kadcyla) has been approved by the U.S. FDA to treat human epidermal growth factor receptor 2 (HER-2)-positive metastatic breast cancer. Despite its effectiveness in most patients, some are initially resistant or develop resistance. No biomarker of drug resistance to T-DM1 has been identified. Antibody-drug efficacy is associated with antibody internalization in the cell; therefore, cellular sensitivity of cells to the drug may be linked to cellular vesicle trafficking systems. Caveolin-1 is a 22 KD protein required for caveolae formation and endocytic membrane transport. In this study, the relationship between caveolin-1 expression and the chemosensitivity of HER-2-positive breast cancer cells to T-DM1 was investigated. Samples from 32 human breast cancer biopsy and normal tissue specimens were evaluated immunohistochemically for caveolin-1 expression. Caveolin-1 was shown to be expressed in 68% (22/32) of the breast cancer specimens. In addition, eight (72.7%, 8/11) HER-2 positive breast cancer specimens had a higher caveolin-1 expression than normal tissues. HER-2-positive BT-474 and SKBR-3 breast cancer cells that express low and moderate levels of caveolin-1, respectively, were treated with trastuzumab or its conjugate T-DM1. Cell viability and molecular localizations of caveolin-1, antibody and its conjugate were examined. Confocal microscopy showed that T-DM1 and caveolin-1 colocalized in SKBR-3 cells, which also were five times more sensitive to the conjugate in terms of cell survival than BT-474 cells, although T-DM1 also showed improved drug efficacy in BT-474 cells than trastuzumab treatment. Caveolin-1 expression in these lines was manipulated by transfection of GFP-tagged caveolin-1 or caveolin-1 siRNA. BT-474 cells overexpressing caveolin-1 were more sensitive to T-DM1 treatment than mock-transfected cells, whereas the siRNA-transfected SKBR-3 cells had decreased sensitivity to T-DM1 than mock-transfected SKBR-3 cells. The expression of caveolin-1 could mediate endocytosis and promote the internalization of T-DM1 into HER-2 positive cancer cells. Thus, caveolin-1 protein may be an effective predictor for determining the outcome of T-DM1 treatment in breast cancer patients.

Trastuzumab emtansine (T-DM1) is an antibody drug conjugate (ADC) that was recently approved for the treatment of HER-2-positive metastatic breast cancer. The drug sensitivity of ADCs depends mainly on the internalization efficiency of the drug. Caveolin-1 was shown to promote T-DM1 internalization and enhance drug sensitivity. Whether caveolin-1 can be overexpressed to improve T-DM1 efficacy is interesting and has the potential for clinical application. In this study, diabetes drug metformin was investigated in terms of induction of caveolin-1 expression for increased efficacy of subsequent T-DM1 application. BT-474 cells were pretreated with metformin, followed by combined therapy with metformin and T-DM1. The T-DM1 internalization and drug efficacy were determined, and the protein expressions for signal transduction were also monitored. Caveolin-1 shRNA was applied to suppress endogenous caveolin-1 expression, and the ability of metformin to promote T-DM1 efficacy was investigated. Result showed that in BT-474 cells pretreated with metformin, cellular caveolin-1 overexpression was induced, which then promoted drug efficacy by enhancing T-DM1 internalization. As cellular caveolin-1 was suppressed by shRNA, the effect of metformin-enhanced T-DM1 cytotoxicity was decreased. This study demonstrated that metformin can be applied prior to T-DM1 treatment to improve the clinical efficacy of T-DM1 by enhancing caveolin-1-mediated endocytosis.

In the process of epithelial mesenchymal transition EMT, the disassembly of junctional adhesion complexes such as E-cadherin is a remarkable sign during changes in cell morphology and polarity. However, E-cadherin expression is dynamic, and is regulated by the cellular endocytic system; it is also involved in cell signaling mechanisms. In this study, we investigated the role of E-cadherin in colorectal tumors and the relationship with recycling endosome protein Rab11 in colon cell transformation. For tissue screening, the expressions of E-cadherin and Rab11 in colorectal tumors were identified by immunohistochemistry in 113 patients with colorectal carcinoma. For the in vitro cell experiment, GFP-tagged Rab11 plasmid was transfected into HT29 colon cells, E-cadherin expression and cell transformation were monitored by Western blot and confocal microscopy. In immunohistochemistry, the mean score of E-cadherin in tumor and normal tissues was 1.41 ± 0.06 and 1.08 ± 0.06 (p < 0.05). The mean score of Rab11 in tumor and normal tissues was 0.51 ± 0.05 and 0.18 ± 0.02 (p < 0.05). Synchronous overexpression of E-cadherin and Rab11 was noted in 74 patients (66.5%) with colorectal carcinoma. When GFP-tagged Rab11 plasmid was overexpressed in cultured colon cell line HT-29, the E-cadherin expression was up-regulated, and cell membrane protrusion was induced, which resulted in cell transformation and cell migration. This study demonstrated the importance of the overexpression of Rab11 and E-cadherin in colorectal cancer. The results indicated that Rab11 together with E-cadherin might be potential markers for colorectal cancer progression and treatment.