Timing is everything: Novel Combinatorial Approaches to Overcome the Immunosuppression in Prostate Cancer

Approaches to Overcome Immunosuppression – Prostate Cancer

Prostate cancer background

The prostate gland is a complex organ roughly the size and shape of a walnut and is located just below the bladder, around the urethra. It is composed of distinct “zones”, the peripheral zone, central zone, transition zone, and the periurethral region. About 70%-80% of prostate cancers originate in the peripheral zone. Cells in the transition zone continues to grow throughout a man’s life, with the tissue around preventing it to grow and expand outward. This constitutes benign prostatic hyperplasia (BPH) and accounts for 20% of prostate cancer.

The main role of the prostate is to produce and secrete prostatic fluid, an essential component of the seminal fluid. Specialized acinar epithelial cells of the peripheral zone of prostate have a unique metabolism characterized by an incomplete Krebs cycle which terminates early with the secretion of citrate, an important component of the prostatic fluid aimed at lubricating, nourishing, and protecting sperm. This tricarboxylic acid (TCA) cycle truncation is caused by the excess of zinc present in the cells which prevents the mitochondrial aconites (ACO2) enzyme to catalyze the oxidation of citrate into isocitrate which is part of the first step in the Krebs cycle. High zinc level has also been found to cause physiological apoptosis.

Therefore, low level of zinc due to either low zinc intake or low uptake caused by the loss of the zinc transporter ZIP1 (Zrt- and Irt-like proteins 1, SLC39A1) contribute to early carcinogenesis events. This is also emphasized by the fact that more than 50% of men over 60, and over 90% of men between 70–80 years of age who suffer from BPH have be found to have low zinc status [1], with 100% of prostate cancer patients having low level of zinc [2]. It might therefore be advisable to ask men over the age of 45 to regularly monitor their zinc level and for those found to have low level to establish the most appropriate way to restore zinc deficiency [3].

ADT and its consequences

Localized prostate cancer has an excellent prognosis due to improved treatments including surgery, radiation, High Intensity Focused Ultrasound (HIFU), and cryosurgery. Of those 30-40% will develop biochemical recurrence (BCR) which will require the use of hormone therapy also referred to as androgen-deprivation therapy (ADT). Following ADT up to 20% will progress to castrate resistant prostate cancer (CRPC) within 5 years of their treatment [4], from which most patients eventually die. Of note, ADT, whether an agonist or an antagonist is nowadays never given alone for mCRPC.

Despite some improvement, progression to castration resistant prostate cancers (CRPC) still represent a major therapeutic challenge for patients with prostate cancer. Notwithstanding this, ADT appears to mitigate T-cell tolerance allowing for T cells priming against prostate specific antigen [5] and significantly increases intratumorally CD8+ T-cell infiltrate, which was shown to be further increased after vaccination. However, ADT was also shown to increase Treg [6]. Overall, many studies have confirmed the effect of ADT on the tumor microenvironment going from a “cold” to pro-inflammatory environment [7,8]. Nonetheless, this local immune activation was found to be transient in mice and seems to disappear with the emergence of castration resistance[5].

Therefore, while ADT opens a short window of opportunity when prostate-specific T-cell are primed, these require to be further “boosted” with the addition of a vaccine and will likely also require a means to reduce/eliminate/convert Tregs and or MDSC. This was also highlighted by the PROSPECT study (cancer vaccine composed of an engineered poxviral vaccine targeting PSA expressing tumor cells given along with a triad of human T-cell costimulatory molecules (B7.1, ICAM-1, and LFA-3, designated TRICOM) [9] which did not meet its primary endpoint, highlighting that cancer vaccines alone are not able to break overcoming immune suppression and triggering protective anti-tumor immunity.

This evidence supports the concept that inducing immunity to prostate-related proteins such as prostatic acid phosphatase (PAP) can be of therapeutic benefit. Sipuleucel-T (PROVENGE™), an immunotherapeutic approach consisting of the in vitro activation of the patient’s own autologous peripheral-blood mononuclear cells with the PAP protein fused to granulocyte-macrophage colony-stimulating factor, PROVENGE™ which has already demonstrated its safety and clinical benefit for the treatment of minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC) and was FDA approved in 2010 [10]. In addition, Anderson et al demonstrated that even after effective initial priming inducing a strong anti-tumor immune response, cells can revert to a tolerant state, which might explain the significant albeit limited clinical efficacy of PROVENGE [11].

Immunosuppressive microenvironment of the prostate and potential interventions

Overall, immune resistance is likely due to low immunogenicity because of the low tumor mutation burden of prostate tumor cells and to an immunosuppressive tumor microenvironment (TME). This limits the efficacy of any active vaccine treatment such as PROVENGE vaccine which remains extremely expensive and therefore not available to everyone. Moreover, while immune checkpoint blockade has changed the outlook for cancer treatment and survival for several tumors since its first approval in 2011; the clinical benefit in CRPC has been limited to patients with mismatch repair deficiency [12]. Several immune-suppressive cells are present in the TMA of prostate cancer, these include tumor associated macrophages (TAM), cancer associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), myeloid derived-suppressor cells (MDSCs) and regulatory T-cells (Tregs) [13]. However out of these MDSC are perhaps the most important ones to target.

Androgen resistance is associated with the recruitment of myeloid-derived suppressor cells (MDSCs) producing IL-23 into the tumor microenvironment (TME) which promotes the survival and proliferation of prostate cancer cells in androgen-deprived conditions[14]. This has been proposed to be driven by local inflammation, stress/depression and obesity which induces neuropeptide Y secretion from prostate cancer cells and MDSC recruitment [15]. Therefore, targeting the immune regulatory properties of MDSC by inhibiting STAT3 or COX2, or prevent their infiltration/activation via targeting EGF, IL-1B, semaphorin 4D or CXCR2, or stop their development/maturation or even by inducing their death[13] needs to be part of the overall immunotherapeutic treatment of prostate cancer, potentially before and during vaccination approaches.

Interestingly, however, a recent study on pre-clinical models of GBM revealed that while anti-PD1 monotherapy did not significantly improve the survival of mice with brain tumor, it was needed to alleviate CD8 T-cell dysfunction as evidenced by increased production of IFNγ which when combined with an agonist GITR antibody was able to convert Treg into Th1 CD4+ T-cells and impacted significantly on the survival of tumor bearing mice[16]. This opens several avenues, one that anti-PD1 might still offers some therapeutic advantages to prostate cancer patients other than those with DNA repairs deficiency, when combined with additional therapies and two that instead of using therapies aimed at removing/deleting Treg one should use strategies that “revert” Treg into effector CD4+ T-cells.

As mentioned before, although most patients with advanced disease initially respond to androgen deprivation therapy, they invariably progress to a castration-resistant state. Paradoxically, the androgen receptor (AR) signaling axis remains intact in castration-resistant prostate cancer (CRPC) [17]. The molecular mechanisms underlying the proliferation of prostate cancer cells under an androgen-deprived environment are currently under investigation. One of these mechanisms involves the covalent acetylation and deacetylation of histone proteins.

Histone deacetylase inhibitors (HDACi) are a group of potent epigenetic drugs which have been investigated for their therapeutic potential in various clinical disorders, including hematological malignancies and solid tumors. Several lines of evidence have shown that HDACs are abundantly expressed and upregulated in prostate cancer [18,19]. HDAC regulates the expression of several functional genes, including the AR in prostate cells as well as have immunoregulatory activity amongst which the inhibition of dendritic cells [20].

Several clinical trials combining HDACi with immunotherapy strategies (mostly checkpoint inhibitors,[21]) are currently ongoing and include the immune checkpoint inhibitor, pembrolizumab in combination with vorinostat for the treatment of advanced renal or urothelial cell carcinoma (ClinicalTrial.gov identifier: NCT02619253). A Phase I Study of Entinostat in combination with enzalutamide for the treatment of patients with castration-resistant prostate cancer is also ongoing (NCT03829930).

Targeting β2-adrenergic receptor

Sympathetic signaling, often associated with obesity and chronic stress, is increasingly acknowledged as a contributor to cancer aggressiveness [22]. In prostate cancer, intact sympathetic nerves are critical for tumor formation, which, when suppressed, induce apoptosis and blocks tumor growth. Interestingly, men who take beta blockers, which interfere with signaling from the stress hormones adrenaline and noradrenaline, have been found to have a lower incidence of prostate cancer [23]. Of particular interest is the fact that most of prostate cancer originate from the peripheral zone which is where most of the nerves are located [24]. Moreover, high risk cancer patients have a higher nerve density than low risk patients (Ayala).

Figure 1 summaries the possible time and interventions for prostate cancer prevention/treatments (Adapted from Shackelton et al. [13]). Proposed sequence of novel combinatorial immunotherapeutic interventions for different stages of PCa.  Black line represents the typical disease progression which ultimately result in death for the majority of mCRPC  patients and arrows indicate therapeutic interventions with  Dotted lines  representing  possible positive outcome after interventions.

Timing is indeed everything, active immunotherapy is likely to be most effective if given shortly after ADT but before prostate adenocarcinoma (PAC) transdifferentiates into an aggressive neuroendocrine prostate cancer (NEPC) (although some can arise de novo) due extensively androgen deprivation therapy (ADT) or radiation treatment(s) [25]. Approximately 25% of men who die of prostate cancer have tumors with a neuroendocrine phenotype.

Neuroendocrine prostate cancer is a poorly defined clinical phenotype of aggressive disease and causes approximately 10–25% of prostate cancer-related deaths [25,26]. NEPC expresses high levels of b2-adrenergic receptors (ADR) which can be activated by adrenergic signals triggered by depression or chronic stress, which is prevalent in men with prostate cancer. Androgen deprivation therapy (ADT) (GNRH agonists or abiraterone) or AR antagonists (bicalutamide or enzalutamide), invariably leads a hormone/castration-resistant state for which there are few treatment options. As mentioned, ADT promotes the development of a neuroendocrine prostate cancer phenotype [27] and increases the prevalence of neuroendocrine cells [28], which are characterized by loss of AR expression, and resistance to hormonal therapies.

However, activation of the β-adrenergic receptor (ADRB) can also induce trans-differentiation of prostate cancer cell lines into neuroendocrine-like cells in vitro [29]. β2-adrenergic receptor (ADRB2) is the most abundant receptor for sympathetic signals in prostate luminal cells. Epidemiologic studies have shown that the use of β-blockers, which inhibit β-adrenergic receptor activity, is associated with a reduced prostate cancer-specific mortality. In addition, meta-analysis has revealed that psychological depression/chronic stress is prevalent in men with prostate cancer [30] and patients with these conditions can deliver increased adrenergic signals via sympathetic nerve fibers, which then act via b-adrenergic receptors (bARs) expressed on cancer cells, thereby promoting cell proliferation.

The majority of laboratory and clinical studies have utilized non-selective β-AR antagonists, such as propranolol, and β2-AR selective antagonists, such as ICI-118,551, to demonstrate the ability of these compounds to block the pro-metastatic influence of both norepinephrine and synthetic analogues. ICI 118551 has previously been compared with placebo and propranolol in a double-blind cross-over trial in 11 chronically anxious patients [31].

However, little work has been undertaken on the design and testing of novel β2-selective beta-blockers. Regardless of the type of β-blockers, some patients will not benefit from such a treatment, thus it is important to first identify patients with an inactive ADRB2 signaling or with active mechanisms that would make ADRB2 blockade inefficient. As such, levels of catecholamines in both plasma and biopsies samples as well as phosphorylation level of PKA substrates namely S133CREB, S157VASP and S75BAD within the tumor microenvironment [32] should first be measured.

Targeting key factors influencing the biology and immunology of prostate cancer and the TME by combining β-adrenergic receptor blockers or HDAC inhibitors with a prostate specific vaccine is an interesting approach which has not been fully tested yet. Indeed, although β-blocker use has been associated with reduced prostate cancer-specific mortality [33,34], it has not yet been combined with both active immunotherapies and HDAC inhibitors.

Conclusion

Prostate cancer is a dynamic and very heterogenous disease which will require approaches to tackle the immunosuppressive tumor microenvironment (TME) as well as approaches to stimulate and maintain an active anti-tumor immune response.  These will have to be delivered in a timely and individualized manner. Moreover, the level of stress/anxiety as well as the overall well-being, including the microbiota of the patient will also dictate when and how many of these interventions the patient can tolerate.

Author’s Affiliation

Stephanie McArdle – John van Geest Cancer Research Centre at Nottingham Trent University; and Centre for Health, Ageing and Understanding Disease, School of Science and Technology, Nottingham Trent University, Nottingham, UK.

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