New paper below.
Another of those 3-4 week interventions prior to radical prostatectomy.
"Genistein significantly altered the expression of four genes, three had established links to cancer cell motility and metastasis."
-Patrick
Full text:
journals.plos.org/plosone/a...
Here is the Discussion section:
"We deployed an upfront screening strategy designed to probe for effects induced by an experimental chemopreventive agent on at risk target tissue in humans. The screen used gene expression profiling, the experimental chemopreventive agent was genistein and the at risk tissue was histologically normal prostate epithelial cells residing in a cancer-containing organ. Through this strategy we demonstrated that genistein selectively modulates genes that regulate cell motility in at risk human prostate tissue.
These findings provide additional support for epidemiological studies that associate dietary genistein with decreased PCa metastasis and mortality [11–16]. They also serve to corroborate prior prospective pre-clinical and clinical studies from a functional standpoint in that current and prior studies demonstrate that genistein inhibits prostate cell motility [1–3, 7, 9, 12]. A point of difference is that in the current study, our unbiased analysis of genistein’s effects on human prostate did not identify MMP-2, or other members of the MEK4 pro-motility pathway. A central reason for this relates to the fact that when screening over 12,000 genes, we built in highly rigorous selection criterion [17]. Our purpose was to reduce false positives. This strategy was successful in that the genes we found altered by genistein in human tissue were confirmed to regulate human PCa cell motility in vitro. We recognize the inherent drawback of our approach is increased false negatives. This is the case with MMP-2. Our prior in vitro findings led us to specifically test MMP-2 in humans, and we did demonstrate in prior reports that it does decrease in prostate tissue after short term treatment of men [2].
Of high importance is the proof-of-principle demonstration that by undertaking an upfront screening strategy in relevant target tissue after prospective treatment of humans with an experimental chemopreventive agent it is possible to rapidly identify a mechanism of action operative in humans relevant to cancer prevention. The potential impact of this is highlighted by considering the fact that for many experimental chemopreventive agents, the literature links each to several different mechanisms of action. As such it is difficult to prioritize the mechanisms that are operative in humans. Multiple factors contribute to this issue, including inherent limitations in preclinical models, use of high concentrations in pre-clinical testing, multiple pharmacologic effects induced by many agents, as well as unknown effects of metabolites.
The current approach offers a relatively efficient pathway for identifying mechanisms operative in human target tissue for experimental chemopreventive agents. As described above we deployed gene arrays, however, this approach could be readily adapted to many other modes of screening. We consider key elements to success to relate to several factors, including choice of target tissue and how that it was harvested. In the current study, we used laser capture microdissection to allow recovery and analysis of a relatively pure population of cells. Further, we focused our analysis on an at risk cell population, in this case normal prostate epithelial cells adjacent to cancer cells. An equally important feature was study design, including choice of agent, dose, randomization, and utilizing a pre-surgical population, thus providing ample high-quality tissue for probing.
A related important concept was that we began with screening approaches in humans, which then informed the design of cell culture-based in vitro studies. In this particular case, we demonstrated that BASP1 suppressed and HCF2 increased cell invasion. While additional studies focusing on these protein products need to be conducted at the pre-clinical level in order to gain a deeper understanding of how they may interface with other identified pathways that regulate cell motility, they can be conducted with the confidence of knowing that they can be pharmacologically modulated in humans. Further, by demonstrating that engineered changes in HCF2 expression mitigated additional pharmacologic efficacy by genistein, we thereby identified a high priority pathway regulating genistein action. Finally, it was important that our in vitro analysis included transformed normal prostate epithelial cell lines, which represent a model of at-risk pre-cancerous cells, and as such, relevant target cells for chemopreventive agents.
A third important achievement in this study relates to a comparative analysis of tissue from Chinese and US men. The differential demographics of cancers across the globe are frequently cited as important learning opportunities. Unfortunately, those opportunities tend to be limited. While the study of incidence and mortality rates across different regions is of very high value for identifying differences in disease occurrence and outcome, the associated confounding factors tend to preclude more definitive statements. Multiple barriers exist when one seeks to acquire and analyze tissue samples across regions for the purpose of examining specific mechanisms of disease. In carrying out the current study, we demonstrated our ability to overcome these barriers. There are many confounding factors that are in play when one just considers a comparison of tissues harvested from men in China to those harvested from men in the US, and this consideration holds true for the current study. It was therefore of high importance that we then used findings from this comparative tissue analysis to inform the design of prospective studies on cells in vitro. The findings from those studies corroborate those from the comparative tissue analysis, and thereby provide data that serves to mitigate the impact of uncontrollable and unknowable confounding variables. These findings notwithstanding, our current findings provide one possible explanation for differences between Chinese and US prostates.
Our comparative analysis of Chinese and US prostate tissue demonstrated decreased levels of MEK4 expression in Chinese prostate compared to that of the US. The magnitude of this decrease was not small, i.e. ~50%, and was statistically significant. This finding was pathway relevant, as MEK4 has been shown to be a pharmacologic target of genistein, and for genistein-mediated inhibition of motility and metastasis in particular [2, 3]. Further, MEK4 has been shown to be a driver of metastasis for human PCa [9]. This finding in Chinese men supports the notion that long-term exposure to genistein leads to down regulation of its pharmacologic target. The concept that long-term exposure leads to compensatory changes is further supported by a consideration of MMP-2 expression. It was increased in Chinese men, compared to US men. This was surprising in that a relatively comprehensive series of studies conducted by us demonstrated that MEK4 drives increases in MMP-2 and in Chinese men, MEK4 was in fact lower compared to US men. Recognizing that all of our prior studies were conducted after short term genistein exposure, we went on to demonstrate that after long term genistein treatment, MMP-2 in fact increases. Together, these findings demonstrate that with prolonged treatment with genistein, cells undergo compensatory changes in pharmacologically relevant target pathways. This results in an altered biomarker response profile. In this particular instance, we demonstrated that this situation is associated with retention of therapeutic efficacy, at least in the models we examined.
There are inherent challenges in ascertaining the biology operative in humans exposed to an agent at low doses over many years. The investigations conveyed in the current manuscript encompass analysis of effects in humans after short-term and long term exposure, build upon a robust set of pre-clinical and clinical studies that provide concordant findings across model systems that span human recombinant protein in vitro, cell line, animal model and prospective human trials, and they encompass analysis after short-term and long-term treatment of human prostate cancer cells in culture. As such, they serve to provide an integrative framework for understanding how an agent with biologically important properties is acting in humans. By demonstrating differences in effects achieved after short term and long term exposure in humans, and then going on to demonstrate those same changes under rigorous cell culture conditions in the laboratory, the current study serves to provide an integrative framework for understanding previous findings, by us and others. Importantly, it also provides a framework for the design of future studies.
Taken together, our findings also raise a cautionary concern for the design of prospective intervention biomarker-based chemoprevention clinical trials. In the field of chemoprevention, it is current practice to conduct short term intervention trials in which central endpoints are putative biomarkers of therapeutic efficacy. The effect of therapy upon such biomarkers is then used to determine further development in a go/no-go type of fashion. Our current findings demonstrate that different effects upon individual biomarkers are observed after short-term exposure to a chemopreventive agent, as compared to that seen after long-term exposure. This appears to be the case with genistein in the current study. In fact, we had previously reported changes in the expression of pathway-relevant proteins in human PCa murine xenografts after several weeks of treatment with dietary amounts of genistein [3]. At that time, we had raised the prospect of long-term compensatory changes. Findings from the current study further support this notion. They raise the prospect that our findings in the particular case of genistein are in fact more generalizable to other experimental chemopreventive agents. This notion is further supported by the fact that compensatory responses to therapeutics for advanced cancer are an expected reality. Given the emphasis placed upon using short term biomarker response information to guide further development of experimental chemopreventive agents, and the associated commitment of resources, findings from the current study provide evidence that this strategy should be closely examined. It will be important for future studies to examine the mechanisms underlying the changes in biomarker responses induced by genistein treatment.
There are inherent limitations to the current study. There were differences between the clinical characteristics between the Chinese and US cohorts. These were statistically significant for age and stage, and trending for Gleason score. This reflects the facts that there are inherent differences in health care delivery between China and the US. It is possible that these factors contributed to differences in molecular profiles identified by us between cohorts. However, an analysis within each cohort for effects by these clinical features upon gene expression failed to yield effects (not shown), thereby serving to mitigate this concern. Another limitation relates to the biological meaning of downregulated MEK4 and upregulated MMP-2 in the Chinese cohort. While we attributed this to mean downregulation of a pro-motility pathway, it is also possible that other compensatory changes within the regulatory pathways counteract this, and the sum effect is nil. Our follow-on prospective in vitro studies of long term genistein treatment provide evidence that therapeutic efficacy is retained.
In summary, we demonstrated that prospective treatment of US men with genistein selectively targeted genes in at risk prostate tissue that regulate human prostate cell motility. Further, the changes in BASP1 and HCF2 expression observed in humans were subsequently shown to regulate cell motility in vitro. These findings supported the value of screening in at-risk tissue after prospective intervention with experimental chemopreventive agents in humans. We then went on to examine pathway-relevant effects in Chinese and US cohorts. These studies demonstrated downregulation of the pharmacologic target of genistein in Chinese men, who experience lifetime exposure to dietary genistein. They also indicate that biomarker expression can change as a function of treatment time."
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