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Olaparib is a pioneering PARP inhibitor (PARPi) approved for treating castration-resistant prostate cancer (CRPC) tumors harboring DNA repair defects, but clinical resistance has been documented. To study acquired resistance, we developed Olaparib-resistant (OlapR) cell lines through chronic Olaparib treatment of LNCaP and C4-2B cell lines. Here, we found that IGFBP3 is highly expressed in acquired (OlapR) and intrinsic (Rv1) models of Olaparib resistance. We show that IGFBP3 expression promotes Olaparib resistance by enhancing DNA repair capacity through activation of EGFR and DNA-PKcs. IGFBP3 depletion enhances efficacy of Olaparib by promoting DNA damage accumulation and subsequently, cell death in resistant models. Mechanistically, we show that silencing IGFBP3 or EGFR expression reduces cell viability and resensitizes OlapR cells to Olaparib treatment. Inhibition of EGFR by Gefitinib suppressed growth of OlapR cells and improved Olaparib sensitivity, thereby phenocopying IGFBP3 inhibition. Collectively, our results highlight IGFBP3 and EGFR as critical mediators of Olaparib resistance.

Current common treatments for castration-resistant prostate cancer (CRPC) typically belong to one of three major categories: next-generation anti-androgen therapies (NGAT) including enzalutamide, abiraterone acetate, apalutamide, and darolutamide; taxane therapy represented by docetaxel; and PARP inhibitors (PARPi) like olaparib. Although these treatments have shown efficacy and have improved outcomes for many patients, some do not survive due to the emergence of therapeutic resistance. The clinical landscape is further complicated by limited knowledge about how the sequence of treatments impacts the development of therapeutic cross-resistance in CRPC. We have developed multiple CRPC models of acquired therapeutic resistance cell sublines from C4-2B cells. These include C4-2B MDVR, C4-2B AbiR, C4-2B ApaR, C4-2B DaroR, TaxR, and 2B-olapR, which are resistant to enzalutamide, abiraterone, apalutamide, darolutamide, docetaxel, and olaparib, respectively. These models are instrumental for analyzing gene expression and assessing responses to various treatments. Our findings reveal distinct cross-resistance characteristics among NGAT-resistant cell sublines. Specifically, resistance to enzalutamide induces resistance to abiraterone and vice versa, while maintaining sensitivity to taxanes and olaparib. Conversely, cells with acquired resistance to docetaxel exhibit cross-resistance to both cabazitaxel and olaparib but retain sensitivity to NGATs like enzalutamide and abiraterone. OlapR cells, significantly resistant to olaparib compared to parental cells, are still responsive to NGATs and docetaxel. Moreover, OlapR models display cross-resistance to other clinically relevant PARP inhibitors, including rucaparib, niraparib, and talazoparib. RNA-sequencing analyses have revealed a complex network of altered gene expressions that influence signaling pathways, energy metabolism, and apoptotic signaling, pivotal to cancer's evolution and progression. The data indicate that resistance mechanisms are distinct among different drug classes. Notably, NGAT-resistant sublines exhibited a significant downregulation of androgen-regulated genes, contrasting to the stable expression noted in olaparib and docetaxel-resistant sublines. These results may have clinical implications by showing that treatments of one class can be sequenced with those from another class, but caution should be taken when sequencing drugs of the same class.

Previous reports have provided evidence that insufficient or excessive maternal folic acid (FA) intake during pregnancy can alter neurodevelopment of the offspring by modulating prenatal neurogenesis. Furthermore, our earlier work in a mouse model confirmed long-term structural changes at the cellular level of either deficient or excessive FA supply by comparably reducing dendritic arborization of cortical projection neurons. Here, we report that excessive amounts of FA decrease arborization of deep layer projection neurons, but not upper layer neurons and that reduced complexity of deep layer neurons is not observed when folic acid is replaced by folinic acid, a stable reduced form of folate. In addition, deficiency of B12, a vitamin that critically regulates folate metabolism, causes even more marked decreases in neuronal arborization in both deep and upper layer neurons and particularly in combination with FA excess. Furthermore, both FA excess and B12 deficiency affect synaptic density and morphology. Our findings point to neurodevelopmental risks associated with insufficient amounts of prenatal B12, particularly in association with high levels of FA intake, suggesting that the neurodevelopmental program is sensitive to an imbalance in the status of these interacting micronutrients.

Resistance to androgen receptor (AR) targeted therapies remains as the main reason for most prostate cancer related deaths. Lineage plasticity resulting in altered, treatment insensitive prostate tumor cell phenotypes such neuroendocrine differentiated prostate cancer is a common manifestation within resistant tumors upon AR-targeted therapies. The mechanisms responsible for lineage plasticity in prostate cancer remain incompletely understood. Here we demonstrate that the enzalutamide resistant MDVR cell line possesses lineage plastic characteristics associated with overexpression of the Wnt transporter Wntless (WLS). Furthermore, we present evidence that overexpression of WLS is common in varying cell line models of lineage plastic prostate cancer, is higher in neuroendocrine patient samples, and positively correlates with the neuroendocrine marker SYP in clinical data. Targeting WLS in lineage plastic cellular models reduces viability and represses lineage plasticity associated gene expression. Our study provides insight into the importance of WLS to the development of lethal resistant prostate cancer and provides a potential target for the treatment of advanced disease.

BackgroundProper cerebral cortical development depends on the tightly orchestrated migration of newly born neurons from the inner ventricular and subventricular zones to the outer cortical plate. Any disturbance in this process during prenatal stages may lead to neuronal migration disorders (NMDs), which can vary in extent from focal to global. Furthermore, NMDs show a substantial comorbidity with other neurodevelopmental disorders, notably autism spectrum disorders (ASDs). Our previous work demonstrated focal neuronal migration defects in mice carrying loss-of-function alleles of the recognized autism risk gene WDFY3. However, the cellular origins of these defects in Wdfy3 mutant mice remain elusive and uncovering it will provide critical insight into WDFY3-dependent disease pathology.MethodsHere, in an effort to untangle the origins of NMDs in Wdfy3lacZ mice, we employed mosaic analysis with double markers (MADM). MADM technology enabled us to genetically distinctly track and phenotypically analyze mutant and wild-type cells concomitantly in vivo using immunofluorescent techniques.ResultsWe revealed a cell autonomous requirement of WDFY3 for accurate laminar positioning of cortical projection neurons and elimination of mispositioned cells during early postnatal life. In addition, we identified significant deviations in dendritic arborization, as well as synaptic density and morphology between wild type, heterozygous, and homozygous Wdfy3 mutant neurons in Wdfy3-MADM reporter mice at postnatal stages.LimitationsWhile Wdfy3 mutant mice have provided valuable insight into prenatal aspects of ASD pathology that remain inaccessible to investigation in humans, like most animal models, they do not a perfectly replicate all aspects of human ASD biology. The lack of human data makes it indeterminate whether morphological deviations described here apply to ASD patients or some of the other neurodevelopmental conditions associated with WDFY3 mutation.ConclusionsOur genetic approach revealed several cell autonomous requirements of WDFY3 in neuronal development that could underlie the pathogenic mechanisms of WDFY3-related neurodevelopmental conditions. The results are also consistent with findings in other ASD animal models and patients and suggest an important role for WDFY3 in regulating neuronal function and interconnectivity in postnatal life.

Folate is an essential micronutrient required for both cellular proliferation through de novo nucleotide synthesis and epigenetic regulation of gene expression through methylation. This dual requirement places a particular demand on folate availability during pregnancy when both rapid cell generation and programmed differentiation of maternal, extraembryonic, and embryonic/fetal tissues are required. Accordingly, prenatal neurodevelopment is particularly susceptible to folate deficiency, which can predispose to neural tube defects, or when effective transport into the brain is impaired, cerebral folate deficiency. Consequently, adequate folate consumption, in the form of folic acid (FA) fortification and supplement use, is widely recommended and has led to a substantial increase in the amount of FA intake during pregnancy in some populations. Here, we show that either maternal folate deficiency or FA excess in mice results in disruptions in folate metabolism of the offspring, suggesting diversion of the folate cycle from methylation to DNA synthesis. Paradoxically, either intervention causes comparable neurodevelopmental changes by delaying prenatal cerebral cortical neurogenesis in favor of late-born neurons. These cytoarchitectural and biochemical alterations are accompanied by behavioral abnormalities in FA test groups compared with controls. Our findings point to overlooked potential neurodevelopmental risks associated with excessively high levels of prenatal FA intake.

Folate is an essential micronutrient required for both cellular proliferation through de novo nucleotide synthesis and epigenetic regulation of gene expression through methylation. This dual requirement places a particular demand on folate availability during pregnancy when both rapid cell generation and programmed differentiation of maternal, extraembryonic, and embryonic/fetal tissues are required. Accordingly, prenatal neurodevelopment is particularly susceptible to folate deficiency, which can predispose to neural tube defects, or when effective transport into the brain is impaired, cerebral folate deficiency. Consequently, adequate folate consumption, in the form of folic acid (FA) fortification and supplement use, is widely recommended and has led to a substantial increase in the amount of FA intake during pregnancy in some populations. Here, we show that either maternal folate deficiency or FA excess in mice results in disruptions in folate metabolism of the offspring, suggesting diversion of the folate cycle from methylation to DNA synthesis. Paradoxically, either intervention causes comparable neurodevelopmental changes by delaying prenatal cerebral cortical neurogenesis in favor of late-born neurons. These cytoarchitectural and biochemical alterations are accompanied by behavioral abnormalities in FA test groups compared with controls. Our findings point to overlooked potential neurodevelopmental risks associated with excessively high levels of prenatal FA intake.

Previous reports have provided evidence that insufficient or excessive maternal folic acid (FA) intake during pregnancy can alter neurodevelopment of the offspring by modulating prenatal neurogenesis. Furthermore, our earlier work in a mouse model confirmed long-term structural changes at the cellular level of either deficient or excessive FA supply by comparably reducing dendritic arborization of cortical projection neurons. Here, we report that excessive amounts of FA decrease arborization of deep layer projection neurons, but not upper layer neurons and that reduced complexity of deep layer neurons is not observed when folic acid is replaced by folinic acid, a stable reduced form of folate. In addition, deficiency of B12, a vitamin that critically regulates folate metabolism, causes even more marked decreases in neuronal arborization in both deep and upper layer neurons and particularly in combination with FA excess. Furthermore, both FA excess and B12 deficiency affect synaptic density and morphology. Our findings point to neurodevelopmental risks associated with insufficient amounts of prenatal B12, particularly in association with high levels of FA intake, suggesting that the neurodevelopmental program is sensitive to an imbalance in the status of these interacting micronutrients. Prenatal folic acid excess and vitamin B12 deficiency are shown to adversely influence neurodevelopment in mice by delaying the generation and migration of late born cerebral cortical neurons and diminishing neuronal dendritic arborization. [ABSTRACT FROM AUTHOR]

The development of resistance to current standard-of-care treatments, such as androgen receptor (AR) targeting therapies, remains a major challenge in the management of advanced prostate cancer. There is an urgent need for therapeutic strategies targeting key resistance drivers, such as AR variants like AR-V7 and steroidogenic enzymes like AKR1C3, to improve outcomes for patients with advanced prostate cancer. Here, we designed, synthesized, and characterized a class of LX compounds targeting both AR/AR variants and AKR1C3. Molecular docking indicated that LX compounds bound to the AKR1C3 active sites. LX1 blocked AKR1C3 enzymatic activity, suppressing the conversion of androstenedione into testosterone. LX compounds also reduced AR/AR-V7 expression and downregulated their target genes. In vitro, LX1 inhibited the growth of prostate cancer cells resistant to antiandrogens, including enzalutamide, abiraterone, apalutamide, and darolutamide. Treatment with LX1 in vivo significantly decreased tumor growth, lowered serum PSA levels, and reduced intratumoral testosterone levels, without affecting mouse body weight. Furthermore, LX1 overcame resistance to enzalutamide treatment, and the combination of LX1 with enzalutamide further suppressed tumor growth. Collectively, the dual effect of LX1 in reducing intratumoral testosterone and AR signaling, along with its synergy with standard therapies in resistant models, underscores its potential as a valuable treatment option for advanced prostate cancer.