Journal: bioRxiv

Article Title: The soluble guanylyl cyclase pathway is inhibited to evade androgen deprivation-induced senescence and enable progression to castration resistance

doi: 10.1101/2023.05.03.537252

Figure Lengend Snippet: Note that all error bars represent ± SEM and * p< 0.05, ** p≤ 0.01, *** p≤ 0.001, **** p≤ 0.0001. A. The α1β1 heterodimer is the predominant form of the soluble guanylyl cyclase (sGC) complex, which binds nitric oxide (NO) to catalytically generate cGMP, which mediates downstream vasodilatory phenomena. B. Heat maps of genes differentially altered in LNCaP SB0 cultured in FBS- vs. CSS-supplemented media (denoted as ‘ADIS Induction’) and LNCaP SB5 vs LNCaP SB0 cultured in CSS-supplemented media (denoted as ‘ADIS Evasion’). Increasing blue intensity signifies increased expression. These analyses indicate the NO-sGC-cGMP signaling reactome is enhanced in the CSPC LNCaP SB0 cells which undergo ADIS, and is downregulated in the emergent CRPC variants, LNCaP SB5, which can proliferate under AD unlike their SB0 counterparts. C. The mRNA levels for GUCY1A1 (α1 subunit gene) and GUCY1B1 (β1 subunit gene) are altered in opposing directions for CSPC SB0 vs. emergent CRPC SB5 cells under AD. Levels were measured by qPCR for the indicated cells and conditions. ActinB was used as the normalization control. Cells were cultured for 5 days in CSS-supplemented media to induce AD. The p-values were established via an unpaired two-tailed Student’s t test. D. Analysis of mRNA levels for GUCY1A1 and GUCY1B1 in prostate tumor samples derived from the TCGA (largely non-metastatic PC) and SU2C (metastatic) public datasets show differences in expression between the two sGC subunits. Transcript levels are expressed as fragments per kilobase of transcript per million (FPKM), and p-values are from an unpaired two-tailed Student’s t test. E. GUCY1A1 and GUCY1B1 mRNA levels across a panel of CSPC and CRPC cell lines (LNCaP SB0, LNCaP SB5, LNAI, 22Rv1) were analyzed by qPCR and indicate stoichiometric dysregulation of the heterodimer. ActinB was used for normalization. Significance was assessed by an unpaired two-tailed Student’s t test. F. Metastatic treatment-resistant PC tumors exhibit non-stoichiometric GUCY1A1 and GUCY1B1 expression within different tumor regions. UMAP plots from publicly available scRNA-seq data are shown (top: only the PC cells are highlighted, middle, bottom: specific sections of the tumor corresponding to the numbered regions (top) are expanded to show the lack of spatial correlation between GUCY1A1- or GUCY1B1- expressing PC cells. G. Dysregulation of sGC subunits is seen at the protein level in CRPC cells. Immunoblotting of total protein lysates (15 µg) from LNCaP SB0, SB5, LNAI and 22Rv1 cells (FBS-supplemented culture) against sGC⍺1 and sGCβ1 with β actin as the loading control. Molecular weights of markers run on the original immunoblot are indicated on the right. H. Lower intratumoral cGMP levels correlate with faster CRPC tumor growth. Intratumoral cGMP levels were measured in flash-frozen tissue from subcutaneous xenograft tumors derived from CSPC LNCaP SB0 cells (n=2), emergent CRPC LNCaP SB5-derived cells (n=4) and fully CRPC LNAI cells (n=4). Endpoint tumor volumes at time of harvest, number of distinct tumors evaluated, and time to palpable CR tumor formation are shown for each category. Significance was assessed by an unpaired two-tailed Student’s t test. I. Trends for sera cGMP levels, pre- vs. post-castration resistance, are shown for matched CSPC/CRPC samples from 10 de-identified PC patients. J. The CSPC vs. CRPC sera cGMP levels from the 10 patients in (I) are shown. Note that patients 25, 42, 45, 71 are deceased; patient 62 had stable disease at comparable time of assessment (see Fig. S1H). Significance was assessed by an unpaired two-tailed Student’s t test.

Article Snippet: Illumina gene expression raw data were transformed using variance- stabilizing transformation (VST) and log2 transformation, and then normalized through quantile normalization using Bioconductor package lumi (v2.24.0).

Techniques: Cell Culture, Expressing, Control, Two Tailed Test, Derivative Assay, Western Blot