Histone Deacetylase Inhibitors as Therapeutics for Endometriosis

Xin Li; Xishi Liu; Sun-Wei Guo

Disclosures

Expert Rev of Obstet Gynecol. 2012;7(5):451-466. 

In This Article

HDACIs as a Promising Therapeutics for Endometriosis

The statement that endometriosis is an epigenetic disease is certainly more than semantics. An epigenetic disease would imply that it could be potentially treated by correcting epigenetic aberrations through pharmacological means.

Thus far, research on pharmacological correction of epigenetic aberrations in endometriosis is confined to the use of HDACIs. This is due to three considerations. First, the overexpression of class I HDACs in endometriosis[42] and adenomyosis[43] would justify the use of HDACIs. Second, DNA methylation and histone modifications are known to work in concert to regulate gene expression,[45,46] although it remains unclear as to whether DNA methylation or histone modification is the primary signal by which gene expression is determined. Therefore, an alteration of histone modification may give rise to a change in DNA methylation, and vice versa. In addition, as indicated by the demethylation of E-cadherin following HDACI treatment, the inhibition of histone deacetylation can lead to DNA demethylation.[37] Third, the focus on HDACIs was also influenced by empirical evidence to suggest that HDACI treatment may be more potent than DMAs in the inhibition of proliferation [Wu Y et al., Unpublished data]. Finally, it was influenced by the fact that the two most widely used DMAs, azacytidine and decitabine, require intravenous or subcutaneous administration for several days.[47,48] This delivery method may be acceptable to cancer patients who are fighting for their lives by any means they can find. However, for a nonfatal disease such as endometriosis or adenomyosis, this can be unacceptable. In addition, early positive results for azacytidine use are undermined by its reported association with profound and prolonged cytopenias and prohibitive gastrointestinal system toxicity.[49] Since drugs for treatment of endometriosis place higher premium on safety profiles,[50] side effects would be a legitimate concern. By contrast, one HDACI – valproic acid (VPA) – was approved by the US FDA over 30 years ago and is associated with a well-known pharmacology and an excellent safety record. As a result of prioritization, the focus was therefore placed on HDACIs as opposed to DMAs. Thus far, published in vitro and in vivo studies on the use of HDACIs as a potential therapeutics for endometriosis have been very encouraging. These findings are summarized in the following sections.

HDACIs as Antiproliferative & Apoptosis-inducing Agents

Treatment of an endometrial stromal cell line with TSA resulted in decreased proliferation.[51] Treatment with TSA or VPA resulted in cell cycle arrest and induction of p21, a cell cycle-related gene.[52] The effect is likely mediated, perhaps in part, by the upregulation of PR-B by TSA,[51] possibly by increasing acetylation of histones in chromatins. These results have been replicated recently by Kawano et al. in primary endometriotic stromal cells using three different classes of HDACIs (VPA, suberoyl anilide bishydroxamine or SAHA and apicidin.)[41] Kawano et al. also show that treatment with HDACIs induced expression of cell cycle-related proteins such as p21, p16, p27 and chk2, as well as apoptosis-related proteins such as cleaved caspase 9 and Bcl-XL, and also elevated acetylation levels in the promoter region of p21, p16, p27 and chk2, as well as acetylated H3 and H4 in endometriotic stromal cells.[41] What is remarkable is that, when it comes to inhibition of proliferation, endometriotic cells are more sensitive to treatment with HDACs than normal endometrial stromal cells,[41,53] a fact that may be further exploited when considering dosing in order to reduce the collateral damage and to improve side-effect profiles.

In an endometriotic epithelial-like cell line, another HDACI, romidepsin, also known as FK-228 and depsipeptide that was originally isolated from a broth culture of Chromobacterium violaceum, has been shown to reduce HDAC activity, induce acetylation of H2A, H2B, H3 and H4, inhibit proliferation and activate apoptosis through induction of p21, caspase 3, caspase 9 and PARP-1, as well as reduction of Cyclin B1 and Cyclin D1.[54]

HDACIs as Antiangiogenic & Anti-inflammatory Agents

HDACIs have been shown to be antiangiogenic and anti-inflammatory in the context of endometriosis. One important player in promoting angiogenesis and inflammation in endometriosis is COX-2 since COX-2 overexpression has been observed in ectopic endometrium,[55] found to correlate with endometriosis-associated pain[56,57] and reported to be a putative biomarker for recurrence.[58] IL-1β-induced COX-2 expression was inhibited by treatment with TSA.[59]

Another important player in suppressing angiogenesis and inflammation in endometriosis is PPARγ. PPARγ agonists have been reported to inhibit VEGF expression and angiogenesis in endometrial cells,[60] inhibit TNF-induced IL-8 production in endometriotic cells[61] and repress ectopic implants in animal models of endometriosis.[62–64] TSA treatment has been shown to upregulate PPARγ expression in endometrial stromal cells.[65]

TSA treatment can also attenuate constitutive and TNFα-induced NF-κB activation in endometriotic cells.[53] Since NF-κB plays pivotal roles in inflammation, proliferation and angiogenesis,[66] and is known to be constitutively activated in endometriosis,[67,68] its attenuation by TSA strongly suggests that HDACIs may be a promising therapeutic agent for endometriosis. In an endometriotic epithelial-like cell line, romidepsin also inhibits the transcription, expression and secretion of VEGF, a known and major factor involved in angiogenesis in endometriosis.[69]

TSA has been observed to inhibit the expression of SLIT2 [Guo S-W et al., Unpublished data], a member of the SLIT family of secretory glycoproteins that have been shown to attract vascular endothelial cells in vitro and promote tumor-induced angiogenesis.[70] This family of genes appear to be a constituent biomarker for recurrence of endometriosis.[71] In a study involving Slit2 transgenic mice, it has been found that Slit2 overexpression significantly increased the microvascular density – a marker of angiogenesis – as well as lesion size.[72]

HDACIs as Invasion-suppressive Agents

HDACIs have also been found to suppress invasion in endometriotic cells. In one stromal-like and one epithelial-like endometriotic cell line, TSA treatment resulted in reactivated E-cadherin expression along with attenuated invasion.[37] This finding suggests that certain cellular phenotypes of endometriotic cells (such as invasiveness) may be epigenetically mediated, and might therefore be resolved by epigenetic reprogramming through pharmaceutical means.

HDACIs as Uterine Contractility-suppressive Agents

Extensive research suggests that uterine dysperistalsis or hyperperistalsis is associated with endometriosis and may be responsible for dysmenorrhea. It has been reported that, compared with women without endometriosis, women with endometriosis have aberrant uterine contractility during menses with elevated frequency, amplitude and basal pressure tone,[73] all of which may greatly enhance the sensation of uterine contractile movement. Coupled with possible uterine hypersensitivity, that is, increased density of sensory nerve fibers in the endometrium and perhaps also in myometrium, the uterine hyperactivity could be easily perceived as pain. It is also reported that there is a lack of synchronization in fundal-cervical contraction in uteri of women complaining of dysmenorrhea.[74] Therefore, normalizing uterine contractility may help to ease dysmenorrhea in women with endometriosis.

HDACIs, such as TSA, suberic bishydroxamate and VPA, have been reported to suppress spontaneous and oxytocin-induced uterine contractility[75] and are likely to rectify uterine dysperistalsis. Interestingly, progesterone, a drug traditionally used for treating endometriosis-associated dysmenorrhea, is also shown to inhibit myometrial contraction.[76] This may explain why progestin therapy is efficacious for treating endometriosis.

HDACIs as Antinociceptive Agents

VPA was initially approved for treating epilepsy. Of clinical importance, its mechanisms of action include increased GABAergic activity, reduction in excitatory neurotransmission and modification of monoamines.[77] It was later approved for treating bipolar disorder and for migraine prophylaxis.[77,78] Several randomized clinical trials indicate that VPA also seems to be efficacious in treating painful neuropathy in Type 2 diabetes.[79,80] VPA and other HDACIs are shown to be efficacious in treating neuropathic pain,[81] inflammatory pain[82] and persistent pain.[83] Therefore, chronic administration of VPA has also been shown to reduce brain N-methyl-D-asparate (NMDA) signaling in rats.[84]

HDACIs as Potent Cytodifferentiation Inducers for Endometrial Glandular Cells: Potential Treatment of Infertility?

Mounting evidence suggest that epigenetic regulation is involved in the physiology and pathological conditions of the endometrium.[44] It is reported that demethylation by a DMA resulted in upregulation of E-cadherin and concomitant increased receptive ability in an (implantation) nonreceptive cell line.[85] In addition, it is reported that the knockdown, by siRNA, of DNMT1, -3A and -3B individually could not upregulate E-cadherin. Knockdown of both DNMT3A and -3B only upregulated E-cadherin partially. Only when all three DNMTs are knocked down simultaneously is E-cadherin completely upregulated.[85] This finding suggests that the uterine receptivity hinges on the downregulation of all three DNMTs.

Treatment of a human endometrial stromal cell line with a DMA for 9 days is reported to result in decidual-like morphology,[86] concomitant with minimal induction of IGFBP-1 and PRL, two well-recognized markers of decidualization. This implicates the involvement of DNMTs, and thus DNA methylation, in endometrial differentiation.

Sakai et al. showed that TSA enhanced the upregulation of IGFBP-1 and PRL in a dose-dependent manner that is directed by E2 + P4 in cultured endometrial stromal cells, but not glandular cells.[87] The co-addition of TSA resulted in the augmentation of morphological changes resembling decidual transformation. They also found that treatment with E2 + P4 significantly increased the levels of acetylated H3 and H4, whose increment was further elevated by co-treatment with TSA. Chromatin immunoprecipitation assay revealed that treatment with E2 + P4 increased the amount of proximal progesterone-responsive region of IGFBP-1 promoter associated with acetylated H4, an effect that was enhanced dramatically by co-administration of TSA. Accordingly, these findings indicate that histone acetylation is intimately involved in the differentiation of human endometrial stromal cells.[87] The same group also reported that Ishikawa cells can be morphologically and functionally differentiated through upregulation of glycodelin by treatment of TSA or SAHA.[88,89] Using an in vitro implantation assay, they also demonstrated that induction of glycodelin following treatment with ovarian steroid hormones or SAHA enhanced implantation.[90] Taken together, their studies indicate that HDACIs may have potential in the treatment of infertility.[90]

Rodent & Human Efficacy Studies of HDACIs in Treating Endometriosis and/or Adenomyosis

Animal studies also show the potential of HDACIs in treating endometriosis. In mice with surgically induced endometriosis, the lesion size is significantly reduced (compared with controls) by treatment with TSA.[91] This finding has also been replicated in rats treated with another HDACI, VPA.[92] More notably, induced endometriosis has been reported to give rise to generalized hyperalgesia or likely 'central sensitization', but TSA or VPA treatment significantly improved rodents' tolerance to invoked pain.[91,92] It is worth noting that the improvement in pain behavior in rats with induced endometriosis is endometriosis specific, not due to the general analgesic property that VPA might have.[92]

Capitalizing on the safety record and the well-known pharmacology of VPA, an HDACI, and also on the fact that adenomyosis, once called endometriosis interna, can be diagnosed quite accurately by ultrasound, the VPA was pilot tested on three patients as a new therapeutic agent and found that it was well tolerated and, after taking it for 2 months, the pain symptoms in women with adenomyosis were dramatically reduced.[93] In addition, uterus size was reduced by an average of one-third. Results from more studies involving 12 patients with symptomatic adenomyosis show that treatment with VPA for 3 months effectively alleviated adenomyosis-associated pain and reduced uterus size by one-fourth.[94] These clinical observations corroborate well with the in vitro data that HDACI treatment suppresses proliferation and cell cycle progression in ectopic endometria in adenomyosis.[24] They are also consistent with the in vivo data that VPA treatment results in a reduction in myometrial infiltration, uterine contractility and contractile irregularity,[95] along with the alleviation of adenomyosis-associated pain.[95,96]

In rats with induced endometriosis, VPA treatment results in significantly decreased immunoreactivity of NMDA receptor 1, calcitonin gene-related peptide, c-Fos, acid-sensing ion channel 3 and TrkA (a high-affinity receptor for NGF) in dorsal root ganglion (DRG), along with improved thermal latency.[97] Since NMDA receptors calcitonin gene-related peptide, c-Fos and ASIC-3 are known to be expressed in sensory neurons in DRG in the presence of central sensitization,[98–100] these results demonstrate that VPA, and perhaps other HDACIs as well, may be efficacious in reducing central sensitization induced by endometriosis and possibly in alleviating endometriosis-associated pain in humans. While these rodent efficacy studies of endometriosis are the first to evaluate potential therapeutic efficacy in relieving endometriosis-induced pain and may be a step further in bridging the gap between basic research and clinical care by aligning outcome measures used in rodent studies with those used in clinical trials, caution should be exercised when interpreting these findings. For one, the pain measured in rodent studies are invoked pain, not spontaneous pain experienced by patients with endometriosis. It is yet to be determined whether the use of the hot plate test or DRG markers is more predictive of future success in clinical trials than just the measurement of lesion size or volume in endometriosis.

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