Transcriptomic and clinical heterogeneity of metastatic disease timing within metastatic castration-sensitive prostate cancer.

BACKGROUND: Metastatic castration-sensitive prostate cancer (mCSPC) is commonly classified into high- and low-volume subgroups which have demonstrated differential biology, prognosis, and response to therapy. Timing of metastasis has similarly demonstrated differences in clinical outcomes; however, less is known about any underlying biologic differences between these disease states. Herein, we aim to compare transcriptomic differences between synchronous and metachronous mCSPC and identify any differential responses to therapy. PATIENTS AND METHODS: We performed an international multi-institutional retrospective review of men with mCSPC who completed RNA expression profiling evaluation of their primary tumor. Patients were stratified according to disease timing (synchronous versus metachronous). The primary endpoint was to identify differences in transcriptomic profiles between disease timing. The median transcriptomic scores between groups were compared with the Mann-Whitney U test. Secondary analyses included determining clinical and transcriptomic variables associated with overall survival (OS) from the time of metastasis. Survival analysis was carried out with the Kaplan-Meier method and multivariable Cox regression. RESULTS: A total of 252 patients were included with a median follow-up of 39.6 months. Patients with synchronous disease experienced worse 5-year OS (39% versus 79%; P < 0.01) and demonstrated lower median androgen receptor (AR) activity (11.78 versus 12.64; P < 0.01) and hallmark androgen response (HAR; 3.15 versus 3.32; P < 0.01). Multivariable Cox regression identified only high-volume disease [hazard ratio (HR) = 4.97, 95% confidence interval (CI) 2.71-9.10; P < 0.01] and HAR score (HR = 0.51, 95% CI 0.28-0.88; P = 0.02) significantly associated with OS. Finally, patients with synchronous (HR = 0.47, 95% CI 0.30-0.72; P < 0.01) but not metachronous (HR = 1.37, 95% CI 0.50-3.92; P = 0.56) disease were found to have better OS with AR and non-AR combination therapy as compared with monotherapy (P value for interaction = 0.05). CONCLUSIONS: We have demonstrated a potential biologic difference between metastatic timing of mCSPC. Specifically, for patients with low-volume disease, those with metachronous low-volume disease have a more hormone-dependent transcriptional profile and exhibit a better prognosis than synchronous low-volume disease.

Individual willingness to leave a safe refuge and the trade-off between food and safety: a test with social fish.

Refuges offer prey animals protection from predation, but increased time spent hiding can reduce foraging opportunities. Within social groups, individuals vary in their refuge use and willingness to forage in the presence of a predator. Here, we examine the relative foraging benefits and mortality costs associated with individual refuge use and foraging behaviour within groups of goldfish (Carassius auratus) under predation risk from an avian predator (little egret-Egretta garzetta). We assessed individual order of emergence from the refuge and participation over 15 group foraging outings, and assigned each fish a daily outing index score. The individual fish that emerged from the refuge earlier than the other group members and that participated in more outings received high outing index scores and consumed more food compared with fish that tended to emerge in posterior positions and participate in fewer outings. However, individual fish that attained high outing index scores suffered a higher risk of predation. Furthermore, the amount of time the egret spent at the pool affected group foraging behaviour: as predation risk increased, groups of fish consumed significantly less food. Our results exemplify the trade-off between foraging success and safety from predation that prey species regularly experience.

Time to revisit? A predator's previous successes and failures in prey capture determine its return time to patches.

In a heterogeneous environment containing multiple patches that may deplete and renew, a forager should be able to detect the quality of food resources within and among patches and choose to exploit them to best maximize returns. From the predator's perspective, the behavioral responses of the prey in a patch will be perceived as depletion when they retreat to refuge and renewal when they reemerge. A predator encountering responsive prey should manage predation risk, and thus behavioral resource depression, by optimally timing its return time to the patch based on prey behavior. We evaluated the foraging decisions of a predator that encountered patches differing in size of the refuge and prey density. We used little egrets and goldfish as predators and prey in an environment that contained three patches (pools). We manipulated prey density and refuge size and availability (using covers) and observed predator foraging behavior. When the egret had previously caught a fish it did not discriminate between the pools, and the return time was similar for all cover types. The fish densities also did not affect the egret decisions to return to pools. However, when it failed to catch fish, it returned sooner to the pool containing the small cover than the larger one. Additionally, after failing to catch fish in patches containing the highest prey density, the egrets subsequently preferred to return to such patches sooner. We show experimentally that previous failures influence the foraging decisions of a predator choosing how quickly to return to a previously visited patch.

State-dependent foraging among social fish in a risky environment.

In the presence of a predator, foraging is a dangerous task. Social individuals can respond to risk by forming groups, benefiting from enhanced collective anti-predator behavior but suffering from increased conspicuousness to predators. Within groups, individuals exhibit variable foraging behavior. One important factor influencing risky foraging behaviour is current energetic state, and individuals must trade off food and safety by deciding when to leave a protected refuge in order to find food. We generated mixed groups of goldfish (Carassius auratus) containing equal numbers of underfed and well-fed individuals and examined individual refuge use and willingness to take risks venturing into risky foraging areas in the presence of an avian predator (little egret-Egretta garzetta). Underfed fish exhibited higher levels of risky behaviour by participating in more foraging outings and emerging from the refuge in frontal group positions, compared with well-fed individuals. As expected, underfed fish benefitted by consuming more food, but surprisingly did not experience higher rates of mortality. This may be due to the fact that the egret predator rarely captured the first fish to emerge from the refuge, preferentially attacked groups of three or more fish, and often captured fish in the chaotic period following a failed initial strike. We demonstrate how differences in energetic condition can influence risk-taking behaviours among social individuals that subsequently influence relative levels of foraging success and group fission-fusion dynamics. Moreover, our results illustrate the risk associated with foraging in larger groups.

LSD1 inhibition suppresses ASCL1 and de-represses YAP1 to drive potent activity against neuroendocrine prostate cancer.

Lysine-specific demethylase 1 (LSD1 or KDM1A ) has emerged as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Among mCRPC subtypes, neuroendocrine prostate cancer (NEPC) is an exceptionally aggressive variant driven by lineage plasticity, an adaptive resistance mechanism to androgen receptor axis-targeted therapies. Our study shows that LSD1 expression is elevated in NEPC and associated with unfavorable clinical outcomes. Using genetic approaches, we validated the on-target effects of LSD1 inhibition across various models. We investigated the therapeutic potential of bomedemstat, an orally bioavailable, irreversible LSD1 inhibitor with low nanomolar potency. Our findings demonstrate potent antitumor activity against CRPC models, including tumor regressions in NEPC patient-derived xenografts. Mechanistically, our study uncovers that LSD1 inhibition suppresses the neuronal transcriptional program by downregulating ASCL1 through disrupting LSD1:INSM1 interactions and de-repressing YAP1 silencing. Our data support the clinical development of LSD1 inhibitors for treating CRPC - especially the aggressive NE phenotype. Statement of Significance: Neuroendocrine prostate cancer presents a clinical challenge due to the lack of effective treatments. Our research demonstrates that bomedemstat, a potent and selective LSD1 inhibitor, effectively combats neuroendocrine prostate cancer by downregulating the ASCL1- dependent NE transcriptional program and re-expressing YAP1.

In prostate cancer needle biopsies, detections of PTEN loss by fluorescence in situ hybridization (FISH) and by immunohistochemistry (IHC) are concordant and show consistent association with upgrading.

The prognostic value of phosphatase and tensin homolog (PTEN) loss in prostate cancer has primarily been evaluated by either fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC). Previously, we found that PTEN loss by IHC was associated with increased risk of upgrading from biopsy (Gleason 3 + 3) to prostatectomy (Gleason 7+). Now, using an evaluable subset of 111 patients with adjacent biopsy sections, we analyzed the association between PTEN deletion in cancer and the odds of upgrading by a highly sensitive and specific four-color FISH assay. We also compared the concordance of PTEN loss by IHC and PTEN deletion by FISH. PTEN deletion was found in 27 % (12/45) of upgraded cases compared with 11 % (7/66) of controls (P = 0.03). Cancers with PTEN deletions were more likely to be upgraded than those without deletions (adjusting for age odds ratio = 3.40, 95 % confidence interval 1.14-10.11). With respect to concordance, of 93 biopsies with PTEN protein detected by IHC, 89 (96 %) had no PTEN deletion by FISH, and of 18 biopsies without PTEN protein by IHC, 15 had homozygous or hemizygous PTEN deletion by FISH. Only 4 biopsies of the 93 (4 %) with PTEN protein intact had PTEN deletion by FISH. When the regions of uncertainty in these biopsies were systematically studied by FISH, intra-tumoral variation of PTEN deletion was found, which could account for variation in immunoreactivity. Thus, FISH provides a different approach to determining PTEN loss when IHC is uncertain. Both FISH and IHC are concordant, showing consistent positive associations between PTEN loss and upgrading.

Cloning and expression in Escherichia coli of the gene encoding beta-C-4-oxygenase, that converts beta-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis.

In the green alga Haematococcus pluvialis the ketocarotenoid astaxanthin accumulates under stress conditions. Astaxanthin is a red carotenoid pigment which possess antioxidative activity. We have cloned the gene for beta-C-4 oxygenase (beta-carotene ketolase) from the green algae H. pluvialis. The cloning method took advantage of a strain of E. coli which was genetically engineered to produce beta-carotene. An expression cDNA library of H. pluvialis was transfected to cells of this strain and visually screened for brown-red pigmented colonies. One colony out of 100,000 transformants showed color change due to accumulation of canthaxanthin. The cDNA clone in this transformant colony encodes the enzyme beta-C-4 oxygenase that catalyzes the conversion of beta carotene to canthaxanthin via echinenone. This enzyme does not convert zeaxanthin to astaxanthin. It is concluded that in H. pluvialis astaxanthin is synthesized via canthaxanthin and therefore an additional enzyme is predicted, which converts canthaxanthin to astaxanthin.

Assessing the order of critical alterations in prostate cancer development and progression by IHC: further evidence that PTEN loss occurs subsequent to ERG gene fusion.

BACKGROUND: ERG rearrangements and PTEN (phosphatase and tensin homolog deleted on chromosome 10) loss are two of the most common genetic alterations in prostate cancer. However, there is still significant controversy regarding the order of events of these two changes during the carcinogenic process. We used immunohistochemistry (IHC) to determine ERG and PTEN status, and calculated the fraction of cases with homogeneous/heterogeneous ERG and PTEN staining in a given tumor. METHODS: Using a single standard tissue section from the index tumor from radical prostatectomies (N=77), enriched for relatively high grade and stage tumors, we examined ERG and PTEN status by IHC. We determined whether ERG or PTEN staining was homogeneous (all tumor cells staining positive) or heterogeneous (focal tumor cell staining) in a given tumor focus. RESULTS: Fifty-seven percent (N=44/77) of tumor foci showed ERG positivity, with 93% of these (N=41/44) cases showing homogeneous ERG staining in which all tumor cells stained positively. Fifty-three percent (N=41/77) of tumor foci showed PTEN loss, and of these 66% (N=27/41) showed heterogeneous PTEN loss. In ERG homogeneously positive cases, any PTEN loss occurred in 56% (N=23/41) of cases, and of these 65% (N=15/23) showed heterogeneous loss. In ERG-negative tumors, 51.5% (N=17/33) showed PTEN loss, and of these 64.7% (N=11/17) showed heterogeneous PTEN loss. In a subset of cases, genomic deletions of PTEN were verified by fluorescence in situ hybridization in regions with PTEN protein loss as compared with regions with intact PTEN protein, which did not show PTEN genomic loss. CONCLUSIONS: These results support the concept that PTEN loss tends to occur as a subclonal event within a given established prostatic carcinoma clone after ERG gene fusion. The combination of ERG and PTEN IHC staining can be used as a simple test to ascertain PTEN and ERG gene rearrangement status within a given prostate cancer in either a research or clinical setting.

Up-regulation of MKK4, MKK6 and MKK7 during prostate cancer progression: an important role for SAPK signalling in prostatic neoplasia.

Identification of the signalling cascades that are differentially activated during prostatic tumourigenesis is a crucial step in the search for future molecular targets in this disease. The stress-activated protein kinase (SAPK) signalling cascade culminates in the phosphorylation of the JNK and p38 mitogen-activated protein kinases (MAPKs). Recently, the upstream activators of these proteins, the MAPK kinases (MKKs), have been implicated as inhibitors of tumour progression in a variety of clinical and experimental tumour models. This study evaluates MKK4, MKK6 and MKK7 expression during prostate cancer progression in humans and in the transgenic adenocarcinoma of a mouse prostate (TRAMP) model of prostate tumourigenesis. Benign prostate, prostatic intraepithelial neoplasia (PIN) lesions and tumour tissues were collected from 37 TRAMP mice. Additionally, six tissue microarrays were constructed with tumours from a matched group of 102 men who underwent radical prostatectomy. Tissues from 20 patients with extensive high-grade prostatic intraepithelial neoplasia (HGPIN) were also analysed. For all samples, immunohistochemical staining for MKK4, MKK6 and MKK7 was scored in normal and neoplastic glands. Staining intensities of MKK4, MKK6 and MKK7 were significantly increased in HGPIN and prostate cancer compared to surrounding normal glands in both the TRAMP and human samples (p < 0.0001 for all markers). Increased levels of MKK4 or MKK7 correlated with higher pathological stage at prostatectomy (p = 0.01 and p = 0.04). Using multivariate analysis, there was no association between protein levels and time to biochemical recurrence in the human samples. The up-regulation of MKK4, MKK6 and MKK7 during prostate cancer progression in both TRAMP and human tissues highlights an important role for the SAPK signalling cascade in prostatic neoplasia. The finding that higher MKK4 and MKK7 expression is associated with higher-stage prostatic tumours underscores the dynamic regulation of these proteins during prostatic tumourigenesis.

Xylanase, a novel elicitor of pathogenesis-related proteins in tobacco, uses a non-ethylene pathway for induction.

Antisera to acidic isoforms of pathogenesis-related proteins were used to measure the induction of these proteins in tobacco (Nicotiana tabacum) leaves. Endo-(1-4)-beta-xylanase purified from culture filtrates of Trichoderma viride was a strong elicitor of pathogenesis-related protein synthesis in tobacco leaves. The synthesis of these proteins was localized to tissue at the area of enzyme application. The inhibitors of ethylene biosynthesis and ethylene action, 1-aminoethoxyvinylglycine and silver thiosulfate, inhibited accumulation of pathogenesis-related proteins induced by tobacco mosaic virus and alpha-aminobutyric acid, but did not inhibit elicitation by xylanase. Likewise, the induction of these proteins by the tobacco pathogen Pseudomonas syringae pv. tabaci was not affected by the inhibitors of ethylene biosynthesis and action. The leaf response to tobacco mosaic virus and alpha-aminobutyric acid was dependent on light in normal and photosynthetically incompetent leaves. In contrast, the response of leaves to xylanase was independent of light. Tobacco mosaic virus and alpha-aminobutyric acid induced concerted accumulation of pathogenesis-related proteins. However, xylanase elicited the accumulation of only a subset of these proteins. Specifically, the plant (1-3)-beta-glucanases, which are normally a part of the concerted response, were underrepresented. These experiments have revealed the presence of a novel ethylene-independent pathway for pathogenesis-related protein induction that is activated by xylanase.

Pathogenesis-related proteins are developmentally regulated in tobacco flowers.

The accumulation of pathogenesis-related proteins (PR) in tobacco leaves has been casually related to pathogen and specific physiological stresses. The known enzymatic function of some of these proteins is potentially antimicrobial. By using antibodies specific to three classes of pathogenesis-related proteins, we examined tobacco plants during their normal growth. The pathogenesis-related proteins accumulated during the normal development of the tobacco flower. The PR-1 class of proteins (biological function unknown) is located in sepal tissue. PR-P, Q polypeptides are endochitinases and are present in pedicels, sepals, anthers, and ovaries. A glycoprotein serologically related to the PR-2,N,O class is a (1,3)-beta-glucanase and is present in pistils. Differential appearance during flower development, in situ localization, and post-translational processing of floral pathogenesis-related proteins point to a hitherto unsuspected function these classes of pathogenesis-related proteins play in the normal process of flowering and reproductive physiology.

A major stylar matrix polypeptide (sp41) is a member of the pathogenesis-related proteins superclass.

A novel stylar-specific glycosylated protein, sp41, was characterized. Sp41 constitutes greater than 12% of the transmitting tract tissue soluble proteins and is mainly localized in the extracellular matrix. Two cDNA clones corresponding to sp41 mRNA were isolated and sequenced. The decoded sequences are, respectively, 80% and 49% homologous to acidic and basic pathogen-induced (1-3)-beta-glucanases of the leaf. Thus a subfamily of (1-3)-beta-glucanase pathogenesis-related (PR) proteins constitutes one of the major stylar matrix proteins. The accumulation of sp41 transcripts in normally developing and elicitor-treated styles and leaves was followed using an RNase protection assay. During development sp41 transcript accumulation starts well after carpel differentiation. It is first detected in styles at 8 days before anthesis. The maximal level of accumulation is reached during anthesis. Elicitor-treated styles do not accumulate the leaf-type (1-3)-beta-glucanase transcript, although they retain the capacity to synthesize leaf-type pathogenesis-related proteins such as the pathogen-induced acidic chitinase. The developmental regulation of sp41 expression points to a role for them in the normal processes of flowering and reproductive physiology.

Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells.

The Arabidopsis LEAFY COTYLEDON1 (LEC1) gene is required for the specification of cotyledon identity and the completion of embryo maturation. We isolated the LEC1 gene and showed that it functions at an early developmental stage to maintain embryonic cell fate. The LEC1 gene encodes a transcription factor homolog, the CCAAT box-binding factor HAP3 subunit. LEC1 RNA accumulates only during seed development in embryo cell types and in endosperm tissue. Ectopic postembryonic expression of the LEC1 gene in vegetative cells induces the expression of embryo-specific genes and initiates formation of embryo-like structures. Our results suggest that LEC1 is an important regulator of embryo development that activates the transcription of genes required for both embryo morphogenesis and cellular differentiation.