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A, Representative images of clonogenic survival assays in CWR22 cells treated with CPCCOEt, VCC185369, and lapatinib alone or in combination with DS-7423 inhibitor. B, Quantification of clonogenic survival assays from A. Results are shown as mean with SE (n = 3; ***, P < 0.001). C, Tumor volume growth curves of CWR22 xenografts for the indicated treatments. Results are shown as mean with SE (n = 4–5; #, P = 0.06; **, P < 0.01). For those individuals that were culled before the end of the experiment due to ethical limits, a growth constant was calculated as (log fold change), and the tumor volume was estimated to obtain the average.

Model proposed describing the PSMA-mGluR1-HER2 mechanism of resistance to dual PI3K-mTOR inhibitor DS-7423 (created with BioRender.com). HER2/X represents the HER2-containing receptor protein complex (X = EGFR or HER3 in Supplementary Fig. S1E and S1F). A feedback mechanism that is activated in response to PI3K/mTOR inhibition involves upregulation of ErbB/HER protein (HER2 or HER3) according to the genetically diverse prostate cancer cell lines we have tested. Downstream effects include a PSMA increase upon DS-7423 treatment that is partially dependent on HER2 signaling. Targeting the PI3K/mTOR pathway has unraveled a complex relationship between PSMA increase and HER2 upregulation via mGluR1 activity (Fig. 4B).

A, qRT-PCR analysis of GRM1 gene expression in CWR22 and 22RV1 cells. Results are shown as mean with SE (n = 3; *, P < 0.05; **, P < 0.01). B, Expression of HER2, HER3, and PSMA in control and GRM1-depleted CWR22 cells. C and D, Quantification of HER2 and PSMA expression in cells from B. Results are shown as mean with SE (n = 3; *, P < 0.05; **, P < 0.01). E, Expression of of HER2, HER3, and PSMA after treatment with inhibitor alone or in combination. F and G, Quantification of expression of HER2 and PSMA presented in E. Results are shown as mean with SE (n = 3).

A, Modulation of expression of PSMA protein in CWR22 and 22RV1 cells by inhibitors with maximum upregulation induced by DS-7423 (3.4- and 5.6-fold, respectively). B, Detection of PSMA mRNA after treatment with DS-7423. Results are shown as mean with SE (n = 3; ***, P < 0.001).

A and C, Expression profile of indicated proteins in CWR22 and 22RV1 cells after treatment with inhibitor alone or in combination. B and D, Quantification of PSMA expression in cells from A and C. Results are shown as mean with SE (n = 3; *, P < 0.05; **, P < 0.01).

Expression profile for indicated proteins in prostate cancer cells after treatment with PI3K, mTOR, or dual inhibitors for 48 hours. A, DS-7423 increased expression of HER2 (4.5-fold in CWR22, 7.3-fold in 22RV1, 1.8-fold in PC3, and 1.2-fold in Shmac5 cells), HER3 (5-fold in LNCap, 1.3-fold in CWR22, 18-fold in PC3, and 4.5-fold in Shmac5 cells), and AR (6.1-fold in LNCaP). B, Immunofluorescence images show the DS-7423–induced upregulation of HER2 (green) and HER3 (red) in CWR22 and 22RV1 cells. Nucleus stained with Hoechst33342 (blue); scale bar, 50 μm.

A new T ₃ P®-mediated multicomponent method was developed for the synthesis of potentially biologically active phosphoryl- or phosphinoyl-functionalized benzo[ de ]isoquinolinones.

Primary open-angle glaucoma remains a global issue, lacking a definitive treatment. Increased intraocular pressure (IOP) is considered the primary risk factor of the disease and it can be caused by fibrotic-like changes in the trabecular meshwork (TM) such as increased tissue stiffness and outflow resistance. Previously, we demonstrated that the sigma-1 receptor (S1R) agonist fluvoxamine (FLU) has anti-fibrotic properties in the kidney and lung. In this study, the localization of the S1R in TM cells was determined, and the anti-fibrotic efficacy of FLU was examined in both mouse and human TM cells. Treatment with FLU reduced the F-actin rearrangement, inhibited cell proliferation and migration induced by the platelet-derived growth factor and decreased the levels of fibrotic proteins. The protective role of the S1R in fibrosis was confirmed by a more pronounced increase in alpha smooth muscle actin and F-actin bundle and clump formation in primary mouse S1R knockout TM cells. Furthermore, FLU demonstrated its protective effects by increasing the production of nitric oxide and facilitating the degradation of the extracellular matrix through the elevation of cathepsin K. These findings suggest that the S1R could be a novel target for the development of anti-fibrotic drugs and offer a new therapeutic approach for glaucoma.

Prostate cancer remains a major cause of male mortality. Genetic alteration of the PI3K/AKT/mTOR pathway is one of the key events in tumor development and progression in prostate cancer, with inactivation of the PTEN tumor suppressor being very common in this cancer type. Extensive evaluation has been performed on the therapeutic potential of PI3K/AKT/mTOR inhibitors and the resistance mechanisms arising in patients with PTEN-mutant background. However, in patients with a PTEN wild-type phenotype, PI3K/AKT/mTOR inhibitors have not demonstrated efficacy, and this remains an area of clinical unmet need. In this study, we have investigated the response of PTEN wild-type prostate cancer cell lines to the dual PI3K/mTOR inhibitor DS-7423 alone or in combination with HER2 inhibitors or mGluR1 inhibitors. Upon treatment with the dual PI3K/mTOR inhibitor DS-7423, PTEN wild-type prostate cancer CWR22/22RV1 cells upregulate expression of the proteins PSMA, mGluR1, and the tyrosine kinase receptor HER2, while PTEN-mutant LNCaP cells upregulate androgen receptor and HER3. PSMA, mGluR1, and HER2 exert control over one another in a positive feedback loop that allows cells to overcome treatment with DS-7423. Concomitant targeting of PI3K/mTOR with either HER2 or mGluR1 inhibitors results in decreased cell survival and tumor growth in xenograft studies. Our results suggest a novel therapeutic possibility for patients with PTEN wild-type PI3K/AKT-mutant prostate cancer based in the combination of PI3K/mTOR blockade with HER2 or mGluR1 inhibitors.

Supplementary Figure from HER2 Mediates PSMA/mGluR1-Driven Resistance to the DS-7423 Dual PI3K/mTOR Inhibitor in PTEN Wild-type Prostate Cancer Models