Updates on Overcoming Bicalutamide Resistance: A Glimpse into Resistance to a Novel Antiandrogen

Abstract

The standard androgen deprivation therapy for advanced prostate cancer includes the use of bicalutamide, which is a well-known antagonist of androgen receptors. Despite numerous benefits of the drugs in prostate cancer treatment, there is always a risk of developing a resistant phenotype, which paves the way for a more aggressive and low-survival type of prostate cancer. Over the years, many studies have investigated the candidate mechanisms of such resistance and have managed to find possible therapeutic solutions. In this Review, we shed light on the heterogeneous dynamics of progression to resistance against bicalutamide treatment, referring to the most recent studies and the approaches that have been so far discussed. This Review tries to offer a deep and comprehensive understanding about how the resistant cells become sensitive to the drug and what corresponding pathways lead to an appropriate solution for the antiandrogen resistance challenge.

After the diagnosis of prostate cancer (PCa), usually, an androgen deprivation therapy (ADT) or maximum androgen blockade treatment is initially offered. Then, castration can be done medically or surgically with antiandrogen drugs to completely inhibit the signaling pathway of the androgen receptor (AR). The first-generation antiandrogens such as bicalutamide, flutamide, cyproterone acetate and, to a lesser extent, nilutamide are the most common drugs used at this stage. Hormone-refractory prostate cancer is a condition with resistance to antiandrogen drugs, while patients show a significant increase in the level of blood PSA despite being on treatment. Nowadays, a second-generation antiandrogen is offered to deal with drug resistance if resistance to the first-generation drugs happens. This strategy targets other regulators in the path of androgen effects, such as using abiraterone that inhibits synthesis and activity of the receptor and ligand complex, which are related to the transcription of the receptors, and this is like what enzalutamide does. However, there are also other methods for dealing with antiandrogen resistance and in particular with bicalutamide resistance including intermittent therapy¹ and bipolar androgen therapy,² in which cycles of drugs at physiological or extra-physiological doses are associated with breaks between treatment courses. It seems crucial to find factors and pathways, which are involved in first-generation antiandrogen resistance, because the use of second-generation antiandrogen drugs can be associated with drawbacks such as limited worldwide availability, occurrence of drug resistance,³ and high prices.

Delaying recurrence of PCa may increase the survival rate and improve the prognosis for some patients. For example, by suppression of known factors of resistance and prolongation of the treatment period with first-generation antiandrogens, it can be possible to impede the progression of hormone refractory that let the castration resistance be postponed too. Accordingly, other treatments such as the second-generation antiandrogens can be avoided as long as the first-generation antiandrogens work. Clinical trials on therapeutic agents acting through the steroid hormone receptor showed that the loss of androgen dependence did not necessarily mean that the tumor became resistant to other hormonal treatments as well.⁴ Therefore, hormone therapy can still be used after the emergence of resistance to flutamide or bicalutamide. Since 1999, investigations on antiandrogen resistance mechanisms, especially bicalutamide, have been explored, and different studies published proposed solutions.⁵ In this Review, all methods that so far have proved to be effective in the battle with bicalutamide resistance are discussed.

AR Modifications and Bicalutamide Resistance

Antiandrogen resistance may root in androgen receptor modifications. Increased expression of the androgen receptor or a mutation in its genomic sequence is known to render tumors capability of resistance to bicalutamide.⁶ However, epigenetic alterations, such as the position of serine phosphorylation within the structure of the AR, have also been shown to play an effective role in the behavior of the receptor in resistant cells. For instance, an increase in the number of AR with phosphorylated serine at position 210 (pAR210) induces bicalutamide resistance through becoming highly sensitive to small amounts of androgen.⁷

Mutation in Structure of AR

Changes in the structure of the AR, caused by genetic mutation, seem to be the main cause of bicalutamide resistance in PCa. Mutations can occur anywhere within the AR-related genome, but those that occur in the binding domain of the androgen receptor appear to have more destructive effects. It has been found that the activity of DNA polymerases switches from a high-fidelity subset to error-prone, and in the meantime, a reduction in expression of proteins involved in the MMR process is the main driver of AR mutations.⁶ This has been shown in resistant cells that bear the W741C/L mutation in comparison with their normal peers. Moreover, the spatial structure of the receptor can be altered by mutation affecting its binding to dihydrotestosterone (DHT), so drugs like bicalutamide, fostering a condition in which bicalutamide acts as an agonist instead of an antagonist,⁸ may cause undesirable consequences such as bicalutamide resistance. The importance of mutation in different positions of the receptor and its presumptive role in converting the receptor from an antagonistic to an agonist response can be investigated by molecular dynamic simulations. Hence, a study using a bicalutamide-resistant cell line followed by androgen-sensitive LNCaP cells being cultured in an androgen-free medium with bicalutamide simulated combined androgen blockade therapy. Over several weeks, these cells grew in the presence of the drug while the prostate specific antigen (PSA) concentration in the flask was increasing. Genomic sequencing of the androgen receptor from these cells showed mutations in the androgen binding site, confirming an antagonist-to-agonist shift had taken place. This study found that exposing these cells to hydroxyflutamide was associated with no changes in binding to the mutant AR, so it maintained its antagonistic behavior and inhibited the activity of receptors.⁹ In the antagonist state, after binding to the receptor, the drug stabilizes the structure of the formed dimers and stops them from moving to the nucleus. In fact, in the presence of a mutation in W741L, the monomers of each dimer are tightly connected, so when reaching the nucleus, they trigger the transcriptional function of the receptor.⁸

It should be noted that antiandrogen drugs affect the selection of mutant forms of AR. A study on biopsy samples from PCa patients who were treated with flutamide or bicalutamide revealed that treatment restricted mutations from gain-of-function, promoter preference, and ligand specificity, which happened as a result of drug pressure. Among the biopsies of hormone-naïve patients, mutations were recurring in areas other than the ligand binding domain (LBD).¹⁰ Resistance to antiandrogen drugs can also occur due to a variety of mutations in AR or other related proteins. Identifying new mutations in relapsed-resistant PCa is highly in the center of attention because biopsies must be taken from bone marrow or deep lymph nodes of the body, which is considered very invasive,¹¹ and DNA sequencing is a good alternative to deal with this challenge.¹² The well-known mutations that play a role in the occurrence of resistance characteristics are listed in Table 1.

Table 1. Mutations with Background of Involvement in Bicalutamide Resistance^a.

mutation	position	type	details11
W741L/C	LBD-741	missense	Involved in bicalutamide resistance phenotype
			Switching from antagonist to agonist in bicalutamide treatment
			Sensitive to Enzalutamide and nilutamide
H874Y	LBD-874	missense	Conversion of antagonist into agonist in the case of flutamide and nilutamide
T877A	LBD-877	missense	Resistance to flutamide and nilutamide
			Flutamide resistance by acquiring the ability of AR binding to other ligands as well

^aLBD: ligand binding domain.

As mentioned above, low AR binding affinity is a major drawback with conventional antiandrogens such as bicalutamide and flutamide. Introducing antiandrogens with different structures may enhance AR binding affinity and has shown potential to overcome bicalutamide resistance. It is known that, even in cases with no dependency on hormone, functional AR is still required,¹³ and chemical studies investigating potential AR inhibitor candidates may pave the way for tackling the clinical conditions of resistance to bicalutamide.

Antiandrogen-Resistant AR Variants Following Alternative Splicing

Multiple splicing patterns of AR mRNA could result in receptors lacking the ligand-binding domain (LBD) and being constitutively active, which are associated with bicalutamide resistance. Among AR variants, androgen receptor variant 7 (AR-V7) is known to function ligand independently and upregulate downstream signaling pathways. Additionally, an increase in the expression of AR-V7 is related to the occurrence of malignant characteristics of PCa and poor prognosis. It is not clear what triggers the shift in AR splicing to produce AR-V7 instead of normal AR, but it seems that its suppressors are downregulated in androgen-resistant tumors. By restoring U2 Small Nuclear RNA Auxiliary Factor 1 expression, which plays a role in the splicing process, AR-V7 production and therefore bicalutamide resistance can be repressed.^14,15

Another study in 2016 found that miR-212, which targets ARV7, was suppressed in bicalutamide-resistant cells, especially in African Americans. In this study, inhibiting a factor involved in the splicing process called hnRNPH1 caused the cytoplasmic AR-V7 concentration to decrease, and the cells became sensitive to bicalutamide. Lin28 has also been seen to reduce the amount of this variant after suppression.¹⁶

Also, two antiparasitic drugs including niclosamide¹⁷ and artesunate¹⁸ are now proven to be effective in suppressing castration-resistant prostate cancer (CRPC) through inhibition of AR-V7 expression.

Increased AR Expression

Many studies show that resistance to antiandrogens occurs only due to an increase in the expression of AR in some patients (compared with the normal state) without the presence of any mutations in the AR sequence. Other studies reported an increase greater than 25-fold in AR expression in CRPC.^2,19 In one study, they continuously exposed LNCaP 104-S cells to bicalutamide and androgen R1881 to produce drug-insensitive cells, and it has been shown that they produce a higher amount of AR, which leads to enhanced AR transcriptional activity.²⁰

Overexpression of AR can also be caused by epigenetic reasons. For example, AR expression is controlled by a repressing sequence in its 5′-UTR region that provides a binding site for the regulatory protein complex. A study claimed that Pur-alpha (Puralpha), a component of the complex, lost its expression in a certain amount of antiandrogen-resistant cells; however, it has been found that its restoration through histone deacetylase inhibitor was effective for sensitizing antiandrogen-resistant cells.²¹

Increased AR expression modifies many aspects of PCa cellular properties, including epigenetic pathways. For example, the DNA methyltransferase (DNMT) enzyme has been reported to be expressed higher in cell lines cultured with bicalutamide. It seems that this increase is regulated to some extent by AR activity because AR inhibition with siRNA can reduce the amount of DNMT. Also, a potent DNMT inhibitor called 5-azacitidine has been found to decrease the bicalutamide resistance in such cells. Based on a clinical study on PCa patients, those with a history of bicalutamide treatment prior to surgery had higher levels of DNMT3a and DNMT3b.^22,23

In addition, one study has claimed that heat shock proteins are involved in bicalutamide resistance via a different mechanism. When an RNA-binding protein called quaking (QKI) increases the concentration of heat shock protein 90, AR consequently becomes activated. As mentioned before, enhanced AR activity is associated with resistance phenotype; therefore, inhibition of QKI protein is capable of increasing cancerous cells’ sensitivity to bicalutamide.²⁴ Contrarily, loss of AR expression or downstream signaling pathway may lead to bicalutamide resistance. It is believed that the explanation lies in cells having no dependency on AR signaling and hypermethylation of the AR gene promoter caused by genetic factors which can block AR expression. If substances with DNMT-inhibitory properties, such as quercetin and curcumin, are simultaneously used on androgen-independent cell lines, then PC3 and DU-145 initiate a general demethylation process that leads to an increase in the AR level of the cells; this in turn results in cells becoming sensitive to DHT. Restoration of normal AR expression within cells is able to stimulate cancerous cells to respond to antiandrogen drugs.²⁵

Bicalutamide-resistant cell lines that are generally established by constitutive increasing doses of the drugs interestingly express a sufficient amount of AR that responds to other first-generation antiandrogens such as hydroxyflutamide. In one study, a combination of flutamide with a chemotherapy agent, 5-fluorouracil, which is an analogue of a pyrimidine base, was capable of inducing apoptosis following an arrest in DNA biosynthesis in bicalutamide-resistant cells. The suppression of downstream genes such as transcription factor E2F1 and thymidylate synthase was identified as the key driver of inhibition of the growth of resistant cells.²⁶

The Role of Cell Membrane and Cytoskeleton Proteins in Bicalutamide Resistance

In addition to alterations in the level of AR expression and its structure, drug resistance can also occur due to transporters that carry drugs out of the cell. This process is common among many types of cancers, while ATP-binding cassettes (ABCs) have been identified as the key transporters. They have numerous subtypes with different roles in each individual solid tumor. Studies on bicalutamide-resistant PCa patients showed that two ABC pumps including P-glycoprotein and breast cancer receptor protein (BCRP) were responsible for the efflux of the drug from the cell in androgen-sensitive LNCaP and androgen-resistant PC3 cells after long-term exposure to the drug. Small molecules inhibiting BCRP and P-glycoprotein decreased the effective dose of bicalutamide by more than half. There was further proof that cells with higher expression of these two pumps could bind to bicalutamide and remove it from the cell.²⁷

In recent years, many studies have proposed unveiling the broad aspects of the mechanisms working behind the bicalutamide resistance. For example, one study revealed that upregulation of a specific isoform of the voltage-dependent calcium channel CaV1.3 caused an increase in store-operated calcium entry after ADT. This mechanism is known to be involved in the occurrence of antiandrogen resistance; therefore, such an oncochannel might be a proper target in handling bicalutamide resistance.²⁸ Additionally, it has been seen that a high expression of protocadherin B9, one of the adhesion proteins located in the prostate cell membrane, is significantly related to poor overall survival. Thus, the role of protocadherin B9 in the progression of PCa can be targeted by designation of a siRNA to reduce the IC50 of bicalutamide to significant levels.²⁹

Moreover, Filamin A is a cytoskeletal protein consisting of several repeats of amino acids plus an actin-binding region. In androgen-dependent LNCaP cells, this protein breaks down and leaves a 90 kDa fragment, which later enters the nucleus and binds to AR. On the contrary, in C4-2 cells, which are independent of androgen, FlnA does not undergo breakdown and remains as one whole in length within the cytoplasm. In these cells, the level of phosphorylated Akt is higher than that in androgen-dependent LNCaP, which can be related to androgen-independent growth. Following the transfection and introduction of this fragment to the nucleus of resistant C4-2 cells, a decrease in the amount of phosphorylated Akt happens and sensitivity to bicalutamide appears in LNCaP. It is known that depletion of FlnA in LNCaP cells with siRNA induces resistance to bicalutamide, and it seems to maintain androgen dependence by regulating the Akt pathway.³⁰ Microtubule-associated protein tau (MAPT), another cytoskeleton component, is found to be responsible for bicalutamide resistance. This protein is regulated by PTEN, which is a component of the Akt pathway and plays a role in tubulin assembly and microtubule stability. It appears that knocking out PTEN can lead to higher amounts of MAPT, so the suppression of MAPT increases the sensitivity to bicalutamide.^31,32

Inhibition of CDK8/CDK19 as cell cycle-promoting proteins shows a synergistic effect on making androgen-independent LNCaP Abl cells sensitized to bicalutamide when simultaneously applied with antiandrogen drugs, and the reason for this can be a decrease in migration capacity, which probably roots in integrin adhesion.³³ Being sensitive to alterations in migration capabilities helps inhibit EMT regulators such as SNAI2 that reduces the malignant properties of the tumor, resulting in drug response. Cytoskeleton components play a key role in the coordination and necessary adjustments for the EMT process.

Disturbances in Signaling Pathways and Cell Metabolism

It is well-known that many signaling pathways undergo alterations to employ malignancy phenotypes in all types of cancer. An increase in the expression profile of signaling components can disrupt the whole pathway and even change the response of cancer cells to the drugs. Although many of such factors are involved in multiple cellular pathways, the keyword of antiandrogen resistance is known to be prominently used with some distinctive pathways. In 2019, based on bioinformatics analysis, a study revealed that bone morphogenetic protein (BMP)-6/SMAD signaling, a known pathway involved in bone metastasis, could be activated during antiandrogen treatment; knowing pathways, which are involved in the development of drug resistance, can assist with the reversal process. β-Catenin signaling is one of the pathways, which is deregulated along with AR signaling in PCa. There is a report that discusses the overactivation of PLCε as a driver of the wnt3a/β-catenin/AR axis in bicalutamide-resistant LNCaP.³⁴ A small-molecule of nuclear β-catenin, called C3, inhibits the use of a β-catenin cofactor, which impedes AR binding to target genes. This event prevents the in vitro formation of bicalutamide-resistant spheres resulting in decreased tumor growth in a xenograft model by inhibition of androgen receptor and β-catenin activity in prostate cancer.³⁵

akt-mTOR is undoubtedly one of the most studied pathways in PCa that together with AR and tyrosine kinase receptors consist of an important pathway in the generation of antiandrogen resistance. Sorafenib, a multiple kinase inhibitor, reduces Akt phosphorylation in bicalutamide/castration-resistant cell lines, making it a suitable candidate for PCa adjuvant therapy in association with antiandrogen drugs.³⁶ High-passage LNCaP cells were used in a study as an in vitro model of bicalutamide resistance. These cells possess a high level of AR expression and bicalutamide insensitivity. However, in this study, bicalutamide suppressed the proliferation of resistant cells by the inhibition of tyrosine kinase receptors (gefitinib) and the mammalian target of rapamycin (everolimus).³⁷

Autophagy is another process related to the androgen-induced mTOR pathway that protects prostate tumor cells against androgen inhibitors and shows an increase by exposure to bicalutamide. Chloroquine, an approved drug for malaria, is an inhibitor of the autophagy pathway that is able to restore bicalutamide sensitivity in cells.^38,39 Calcium/calmodulin-dependent protein kinase II has a downregulated expression level in PCa, and several studies have shown that this protein has a tumor suppressor, which plays a role in regulating processes such as the cell cycle, apoptosis, and AR expression. The AR pathway suppresses CAMK2N1, and this negative self-regulation loop is triggered by the AKT pathway. Engineered cells with a higher level of this protein show more sensitivity to bicalutamide.⁴⁰

In general, understanding the mechanism of inhibition for each first-generation antiandrogen drug on AR function can be useful in designing a treatment regimen. For example, by using bicalutamide and hydroxyflutamide, a significant part of AR is transferred to the nucleus, but it is prevented from transcriptional activity due to corepressor proteins that connect to the receptor–drug complex, which ultimately inhibits the drug’s effect. Hey1 is one of the proposed proteins as acting as a corepressor of the receptor–drug complex and is involved in the Notch pathway. This protein specifically inhibits the ligand-dependent activity of the androgen receptor, making it a distinct therapeutic target.⁴¹ In another study targeting CSL, which is a transcription factor of the Notch pathway, the sensitivity of resistant cells to bicalutamide increased. CSL actually regulates AKT, which can affect AR expression.⁴²

NF-κB is another well-studied pathway in bicalutamide-resistant tumors. Hexamethylene bis-acetamide inducible protein 1 is a nuclear protein involved in the NF-κB signaling pathway and binds to the AR molecule to inhibit its function. Data from the Oncomine database show that the expression of HEXIM1 decreases with the progression of PCa and resistance to hormone therapy at the mRNA and protein levels. It was seen that the ectopic expression of this protein in castration-resistant C4-2 cells increased their sensitivity to bicalutamide. In fact, HEXIM1 inhibits methylation of histone by inducing the expression of histone demethylase KDM5B. This in turn inhibits FOXA1 activity (forkhead box A1), which is directly implicated in shaping signaling by AR.⁴³ One of the impacts of the NF-κB pathway on AR is the increase in the expression of splicing variants of the AR gene. In general, it seems that this pathway plays a significant role in directing AR-mRNA splicing toward the production of resistant variants such as AR-V7 through the c-Myc:Lin28 arm, making cancerous cells unresponsive to bicalutamide.⁴⁴ A study showed that an inhibitor of casein kinase 2 (CK2) protein was able to sensitize cancerous cells to bicalutamide by reducing the amount of AR-V7. Since CK2 is a key regulator in phosphorylation patterns of the NF-κB pathway, this proves the existence of a link between the NF-κB pathway and the expression of AR-V7.⁴⁵ Also, in human prostate tumor xenograft models, inhibiting the NF-κB pathway together with bicalutamide effectively declined tumor growth.⁴⁶

In 2021, another study showed increased expression of the neuroendocrine (NE) hormone gastrin releasing peptide (GRP) and its receptor on the surface of prostate cells could ensue long-term ADTeth. Subsequently, NF-κB pathway inhibition and AR-V7 transcript expression faced decreased efficiency.⁴⁷

In addition to disruptions that happen in cell signaling, it is believed that metabolism reprogramming is taking part in the bicalutamide-resistant state too. A study in 2021 indicated that Terbinafine, an antifungal medicine, could sensitize bicalutamide-resistant cells via altering cholesterol metabolism.⁴⁸ In another study, Carbidopa, the inhibitor of l-dopa decarboxylase enzyme, was used in combination with bicalutamide on a PCa xenograft model, and the result was associated with a decreased tumor growth by 84.4% compared to the control group. This reveals the importance of enzymes in cellular metabolism regarding antiandrogen resistance. Fatty acid metabolism is increased by the progression of PCa, which activates long-chain fatty acyl-CoA synthetase 4 (ACSL4) and is significantly higher in castration-resistant cells compared to hormone-naive cells. Cells that ectopically expressed this enzyme also showed resistance to bicalutamide, so targeting ACSL4 can increase drug efficacy and overcome antiandrogen resistance.^49,50

A group of miRNAs faces decreased expression when a tumor develops. Most of these miRNAs play the role of tumor suppression; thus, restoration of their expression inhibits tumor growth, which also applies to antiandrogen resistance. Some miRNAs have an effect on the drugs, when their expression is suppressed during treatment. First, identification of these miRNAs and then restoration of their expression can remove the resistance to antiandrogen drugs. For example, miR-137 that is suppressed in many cancers leads to uncontrollable growth of the tumor and, in fact, targets a protein called tripartite motif-containing 24 (TRIM24). This protein is involved in stopping tumor cell division and glutamine metabolism. There was a gene therapy trial aimed to restore miR-137 or inhibit TRIM24 and managed to increase cell sensitivity to bicalutamide.⁵¹

Despite numerous efforts to understand and control bicalutamide resistance in PCa cells, there is still a lack of conclusive evidence of the universality of these alterations. For example, no study could demonstrate a consistent correlation between disrupted akt-mTOR signaling and increased cell adhesion molecules or imbalanced microRNA patterns. Thus far, it can only be concluded that targeting individual factors can be efficient in some certain cases. It is important to acknowledge that the function of the AR signaling pathway in PCa cells can act as a double-edged sword. This is because both overexpression and downregulation of AR signaling have been found to promote the development of a resistant phenotype against castration and bicalutamide treatment.

Furthermore, a disparity exists between resistant PCa cells in the laboratory studies and bicalutamide-resistant prostate tissue observed in actual patients. Identifying factors involved in drug resistance can be like a puzzle, with each piece contributing to the discovery of a key regulator in the future. It is important to note that these studies are of great value because they detect prognostic factors. For instance, the involvement of BMP6/SMAD in bicalutamide resistance can be used together with phospholipase C as a marker for poor prognosis in patients with prostate cancer that highlights the potential clinical implications of such findings.³⁴ All key molecular pathways in bicalutamide resistance and their protein crosstalk are presented in Figure 1.

Figure 1.

RTK, pcdh-B9, LRP/Wnt, Notch, Cytokines, and GRPR as the key receptor molecules that trigger bicalutamide resistance and their protein cross talk. Cytoskeleton, cell signaling components as therapeutic targets in overcoming bicalutamide resistance.

The cell adhesion protein pcdh B9 and whole length FlnA are associated with resistance phenotype. Microtubule-associated protein tau (MAPT) that is regulated by PTEN, a component of the Akt pathway, may reduce drug sensitivity. Inhibition of CDK8/CDK19 alters cellular adhesion capacity in PCa and promotes a malignant state. Transcription factor of Notch signaling CSL induces genes that further regulate the AR activity. An increased amount of ß-catenin, a component of WNT signaling in PCa cells, manipulates the transcription activities of AR. HEXIM-1, a nuclear protein involved in the NF-kB signaling pathway, binds to the AR molecule and inhibits prospect function. Following activation of the NF-κB pathway, the amount of ligand-independent variant of AR-V7 is reduced. This transcript is also suppressed by the gastrin releasing peptide (GRP) receptor antagonist, which is increased in drug-resistant cancerous cells of prostate. Autophagy is another way of helping cells escape from bicalutamide and acquire a resistant phenotype.

Tumor Microenvironment and Bicalutamide Resistance

Currently, the standard treatment for advanced PCa is ADT, which can be combined with nonsteroidal antiandrogens. Although maximum androgen blockade is ideally effective in many patients, eventually, it may lead to resistance. Interleukin 6 (IL-6) is among the cytokines that are involved in the occurrence of malignant characteristics of PCa and plays a role in resistance to bicalutamide and progression to the CRPC stage too. Increased production of IL-6 by bicalutamide-treated LNCaP cells can be a result of overexpression of CYP1B1, an enzyme involved in the conversion of estrogen steroid hormone. Prostate cancer stem cells manifest castration- and bicalutamide-resistant phenotypes through IL6-STAT3 signaling, so knocking down the enzyme can reverse the process.⁵² Therefore, targeting IL-6 together with bicalutamide administration is suggested to provide a more effective treatment. IL-6 acts through transcriptional intermediary factor 2 (TIF2) in PCa cells, and it appears to be the corresponding pathway for bicalutamide resistance as well. It showed resistance to bicalutamide when high-passage LNCaP cells are exposed to IL-6 for a long time, and the level of transcriptional intermediary factor 2 (TIF2) is significantly higher than in the parental cells. Targeting TIF2 by using short hairpin RNA may increase the bicalutamide sensitivity among resistant cells.⁵³

The production of nitric oxide as a component exists in the tumor microenvironment and is increased by overexpression of nitric oxide synthase (NOS) enzyme within endothelial cells of hormone-refractory prostate cancer tissues. It causes a decrease in AR activity, so when treating cells with L-NAME that is an inhibitor of NOS, the resistant cells again will respond to bicalutamide.⁵⁴

It has been found that the chemical factors involved in chronic inflammation can also play a role in the occurrence of bicalutamide resistance. Some studies showed that cells under continuous treatment with interleukin-1β, a cytokine associated with inflammation, initially responded to such a stimulus via decreasing AR expression; although after several passages, AR expression was restored even in the absence of interleukin-1β.⁵⁵

Library-based screening studies on cells that became bicalutamide-resistant following repeated treatment during a considerable period of time revealed that miRNAs were involved in the resistance process to some extent. For example, miR-216a, which is induced by DHT present in the microenvironment around the tumor, is increased in such cells, and this has been confirmed by comparison of the microarray data set between cancerous and benign tissues.⁵⁶

There is also a report referring to CAFs (cancer-associated fibroblasts) as a key driver of antiandrogen resistance. Neuregulin 1 that is released by these cells promotes a resistant phenotype through the NRG1/HER3 axis; thus, many studies are targeting the inhibiting factors for clinical use in such situations.⁵⁷

Second-Line Hormone Therapy

Despite the fact that, in the clinical guidelines for PCa, alternative antiandrogen therapy, which means switching from one first-generation antiandrogen to another, is no longer recommended (due to the recent introduction of more effective drugs), these treatments are believed to still be effective in many patients with a resistance background.⁵⁸ As an example, in 2019, there was been a report on using flutamide to control PCa for 10 years after the resistance to bicalutamide happened.⁵⁹ One of the advantages of this method is its affordable cost, which is significant compared to that of the expensive newly developed counterparts. In addition, this treatment seems to be safe and have no serious side effects.⁶⁰ In another study, patients who had a history of treatment with maximum androgen blockade (MAB) using bicalutamide (80 mg/day) were switched to flutamide as the second-line agent (375 mg/day) after the relapse, and changes in PSA were studied as an indicator to evaluate the treatment. There were nine out of 13 patients with a PSA value less than half of the initial value and considered as responders to the treatment. This reduction was maintained for an average period of 11 months. It was also observed that patients who responded to the first-line MAB treatment for a longer period showed a more significant response to this treatment. Therefore, flutamide as a second-line agent seems to be an effective candidate in patients with a background of using MAB bicaltamide.⁶¹

Moreover, in 2010, flutamide was used in patients with CRPC who were initially treated by bicalutamide. This treatment showed a better response in terms of PSA reduction and duration of response among patients who had higher levels of androstenediol and lower levels of dehydroepiandrosterone. This study claimed that the amount of adrenal hormones in the blood potentially played a role in the patient’s response to hormonal treatment of PCa.⁶² Furthermore, treatment with flutamide as a secondary alternative antiandrogen therapy indicates that the initial PSA, T and N stages, and the length of the previous response to the combined androgen blockade treatment are the most important influencing factors on the survival of patients with advanced PCa.

Similarly, an investigation on nilutamide, which is a second-line hormonal agent, showed that patients with a history of treatment by bicalutamide alone or in combination with flutamide would respond to nilutamide and be associated with a drop in blood PSA levels. The effect of this treatment could last up to 11 months.⁶³ However, another study on 28 patients between 1998 and 2001 used nilutamide after resistance to flutamide or bicalutamide and concluded that the reduction of PSA could last up to 21 months.⁶⁴

One of the drawbacks of such studies is the lack of using multiple criteria for evaluation of the patient’s response to treatment. In most of them, only a decrease in blood PSA value was monitored as the sole indicator of response. Nevertheless, their results are always associated with a range of diversity in response to treatment when assessed on the dropping rate of blood PSA. This variation can also be caused by the patient’s genetic background; therefore, with now increasing awareness of personalized medicine, shifting from one specific antiandrogen to another may be considered to magnify the effectiveness of treatment in distinctive groups of patients.

In addition to employing other antiandrogen drugs, some physicians consider periodic treatment with cycles of drug administration followed by intervals of a withdrawal period to tackle the possibility of drug resistance. There is evidence showing that this method initially postpones the development of resistance. Intermittent androgen deprivation, in which patients receive cycles of treatment and then a gap with no antiandrogen, has proved to be effective in patients who suffer from the bicalutamide-resistant phenotype and has managed to improve the outcomes for different stages of PCa with a reduction in mortality rate.

Conclusion and Future Perspective

This Review provides a comprehensive review of the authentic studies so far in the field that have addressed resistance to bicalutamide, which is a first-generation drug for the treatment of prostate cancer. However, there were some successes in both in vitro and in vivo stages; only a limited number of these studies have reached the stage of a clinical trial. Nonetheless, it is imperative to identify the underlying pathways and responsible factors that cause resistance states against bicalutamide treatment to find more effective treatment in the future. Such studies, of course, come with their own challenges. One challenge lies in the use of in vitro resistance simulation models, which often employ cell lines that may not accurately reflect the development of drug resistance in actual patients.

It is believed that continuous exposure of cells to multiple sublethal doses (IC50) may be a better model for studying progression of drug resistance. Additionally, the methods of manipulation employed in these studies are another challenge to overcome. Many of the approaches that have been used to sensitize cancerous cells to the drugs are not yet approved for clinical use. Therefore, even with the identification of resistance-causing factors, still bringing the achieved knowledge to the patient’s bed requires progress in other areas of sciences such as drug delivery.

Consequently, a primary objective of current research is to utilize relevant drugs to target resistance factors and repurpose them in combination with bicalutamide to delay the onset of resistance, followed by suppressing its progression through manipulation of different pathways, in terms of preventing metastasis. For example Artesunate, which is a FDA-approved drug for malaria, showed great promise on controlling prostate tumor in some preclinical studies.¹⁸ However, it is important to note that signaling pathways interact with one another and can have a vast range of effects on outcome. It is assumed that still further studies are required to understand the impact of inhibiting capability of one pathway on other tumoral behaviors, such as the epithelial-mesenchymal transition (EMT).

It should be kept in mind that drug-resistant cells can be targeted by a different kind of treatment, along with administration of bicalutamide. For instance, a clinical trial using a combination of mTOR inhibitor (everolimus) with bicalutamide showed a significant effect on nearly 75% of CRPC bicalutamide-naïve patients (decreased PSA level in 18 out of 24 patients) in comparison with previous trials; it can be assumed that mTOR inhibition may be beneficial as far as drug resistance has not yet developed. This allows the bicalutamide plus everolimus regimen to be considered as a suitable choice at the initial stage of treatment; however, a phase 3 trial is required to confirm this hypothesis.⁶⁵

We believe that future studies will shed more light on the clinical effectiveness of the factors discussed in this Review. We suggest that some of these factors may not be directly related to the occurrence of bicalutamide resistance, but the patient’s genetic background plays the mail role as different occurrence rates of resistance in different patients can explain the possibility of genetic and epigenetic effects. The expansion of our knowledge in the field of personalized medicine allows for the development of tailored treatment regimens for each individual patient, which is supposed to minimize drug toxicity by avoiding inappropriate drugs for that individual patient’s condition.

Glossary

Abbreviations

PCa

Prostate Cancer

AR

Androgen Receptor

ADT

Androgen Deprivation Therapy

PSA

Prostate Specific Antigen

DHT

Dihydrotestosterone

Author Contributions

S.M.K.A. is the principal investigator. M.I. and F.K. collected data. M.I. wrote the manuscript. Z.A. provided the figures based on the text, and H.R. edited the manuscript.

The authors declare no competing financial interest.

Notes

Mailing Address: (S.M.K.A.) Urology Research Center, Sina Hospital, Hassan Abad Sq., Imam Khomeini Ave., Tehran, Iran.