Differential regulation of human monocytes and NK cells by antibody-opsonized tumors.

The monocyte network is important for therapeutic efficacy of antibody therapies against cancer. One mechanism which monocytes/macrophages use to kill cancer cells is phagocytosis. Using trastuzumab and human breast cancer cell lines as a model, we used flow cytometry to evaluate the importance of avidity, antigen density, Fcγ receptor (FcγR) expression, and FcγR polymorphisms in human monocyte phagocytosis. By increasing avidity for the tumor through the addition of pertuzumab to trastuzumab, there was a two-to-threefold increase in phagocytosis potency against the HCC1419 cell line compared to antibodies alone, while NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) failed to increase tumor cell death. Consistent with increasing the avidity through multiple antibodies, antigen density significantly enhanced phagocytosis with breast cancer cell lines that were HER2 gene-amplified compared to non-amplified tumor cells. Confirmation that high antigen density enhanced phagocytosis was obtained when HER2 was overexpressed in HER2 non-amplified cell lines. In contrast, NK cell ADCC failed to distinguish differences in tumor cell death when comparing gene-amplified and non-amplified breast cancer cell lines. The level of phagocytosis was influenced by FcγRIIa and FcγRIIIa expression. Most monocytes are FcγRIIIa-, and the induction of the receptor significantly enhances antibody-dependent phagocytosis. Although both receptors are involved, when blocked FcγRIIIa had a greater influence on phagocytosis. Furthermore, the polymorphism FcγRIIIa 158V significantly enhanced phagocytosis; whereas FcγRIIa 131H polymorphism appeared to improve phagocytosis but was not statistically significant. Targeting of monocytes for enhanced phagocytosis may improve the effectiveness of therapeutic antibodies to improve clinical outcomes.

WEE1 inhibitor, AZD1775, overcomes trastuzumab resistance by targeting cancer stem-like properties in HER2-positive breast cancer.

Although trastuzumab has greatly improved the outcome of HER2-positive breast cancer, the emergence of resistance hampers its clinical benefits. Trastuzumab resistance is a multi-factorial consequence predominantly due to presence of cancer stem-like cells (CSCs). AZD1775, a potent anti-cancer agent targeting WEE1 kinase to drive tumor cells with DNA damage to premature mitosis, has previously shown high efficacies when targeting different cancers with a well-tolerated cytotoxic profile, but has not been evaluated in trastuzumab-resistant (TrR) breast cancer. We sought to investigate the effect of AZD1775 on cancer stem-like cell (CSC) properties, apoptosis, cell cycle regulation in TrR breast cancer. Our study for the first time demonstrated that AZD1775 induces apoptosis and arrests TrR cells at G2/M phase. More importantly, AZD1775 effectively targeted CSC properties by suppressing MUC1 expression levels. AZD1775 administration also induced apoptosis in our in-house patient-derived tumor cell line at passage 0, implying its significant clinical relevance. These findings highlight the potential clinical application of AZD1775 in overcoming trastuzumab resistance in breast cancer.

Generation of unique poliovirus RNA replication organelles.

Poliovirus (PV), a model for interactions of picornaviruses with host cells, replicates its genomic RNA in association with cellular membranes. The origin of PV replication membranes has not been determined. Hypotheses about the origin of replication membranes, based largely on localization of viral proteins, include modification of coat protein complex I (COPI) and/or COPII secretory pathway vesicles and subversion of autophagic membranes. Here, we use an antibody against double-stranded RNA (dsRNA) to identify replication complexes by detection of dsRNA replication intermediates. dsRNA signal is dependent on virus genome replication and colocalizes with the viral integral membrane protein 3A, which is part of the RNA replication complex. We show that early in infection, dsRNA does not colocalize with a marker for autophagic vesicles, making it unlikely that autophagosomes contribute to the generation of PV RNA replication membranes. We also find that dsRNA does not colocalize with a marker of the COPII coat, Sec31, and, in fact, we demonstrate proteasome-dependent loss of full-length Sec31 during PV infection. These data indicate that COPII vesicles are an unlikely source of PV replication membranes. We show that the Golgi resident G-protein Arf1 and its associated guanine nucleotide exchange factor (GEF), GBF1, transiently colocalize with dsRNA early in infection. In uninfected cells, Arf1 nucleates COPI coat formation, although during infection the COPI coat itself does not colocalize with dsRNA. Phosphatidylinositol-4-phosphate, which is associated with enterovirus-induced vesicles, tightly colocalizes with Arf1/GBF1 throughout infection. Our data point to a noncanonical role for some of the COPI-generating machinery in producing unique replication surfaces for PV RNA replication. IMPORTANCE Picornaviruses are a diverse and major cause of human disease, and their genomes replicate in association with intracellular membranes. There are multiple hypotheses to explain the nature and origin of these membranes, and a complete understanding of the host requirements for membrane rearrangement would provide novel drug targets essential for viral genome replication. Here, we study the model picornavirus, poliovirus, and show that some, but not all, components of the cellular machinery required for retrograde traffic from the Golgi apparatus to the endoplasmic reticulum are transiently present at the sites of viral RNA replication. We also show that the full-length Sec31 protein, which has been suggested to be present on PV RNA replication membranes, is lost during infection in a proteasome-dependent manner. This study helps to reconcile multiple hypotheses about the origin of poliovirus replication membranes and points to known host cell protein complexes that would make likely drug targets to inhibit picornavirus infections.