Genetic Predisposition and Chemotherapeutic Approaches Including Oncolytic Virus for the Treatment of Prostatic Adenocarcinoma

Among the leading causes of cancer mortality, prostatic adenocarcinoma (PaC) is at second to lung carcinoma, but it is the most commonly happening non-cutaneous malignancy in elderly men in the world. Therapeutic options for PaC depend on age, growth & stage of malignancy, the desired outcomes and shortcomings of available treatment, estimated cost and patient compliance. Patients older than 60 years with a sluggish localized tumor may be placed on active surveillance, otherwise go with transurethral resection of the prostate (TURP), prostate artery embolization (PAE) and pelvic lymphadenectomy with/without radiation therapy. For metastatic PC androgen-deprivation therapy is an option with or without surgery. These agents decline the body’s testosterone production or block its activity by gonadotropinreleasing hormone (GnRH) analogues including leuprolide acetate and goserelin acetate implant. The hormone’s activity can be stopped by androgens antagonist such as flutamide, bicalutamide and nilutamide along with chemotherapeutic agents, such as taxanes (e.g., docetaxel, paclitaxel) but after all the disease relapses in 20-30% of patients. So, new immunological or vaccine-based therapeutic moieties have been investigated to meet the objective of providing selectivity to cancerous cells and desired therapeutic outcomes with less/no harmful effects to normal cells. The chimeric version, oncolytic poliovirus and human rhinovirus i.e. PVSRIPO is most promising feature in cancer therapeutics and activate innate immunity by neutrophils infiltration via PAMP & DAMP pathways while Sipuleucel-T expresses major histocompatibility complex (MHC) which can stimulate CD4+ helper T-cells and CD8+ cytotoxic T-cells and ultimately activate the acquired immunity against cancer cells. In this article, we have discussed the role of genetic predisposition and chemotherapeutic approaches including Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 September 2021 doi:10.20944/preprints202109.0366.v1 © 2021 by the author(s). Distributed under a Creative Commons CC BY license. oncolytic poliovirus for the treatment of PaC in order to better understanding of tumor biology and mechanisms involved in chemotherapeutic drugs based resistance.

: According to Gleason Pattern scale, lower grade (stage 1, score 1, 2) shows cancer is in initial phase, slow growing and not aggressive. From stage 2-5 and score 3-7 shows cancer is growing rapidly towards bones, lymph nodes and blood vessels (7).
If the disease is considerably alleviated or not responsive to ADT referred as Castration Sensitive Prostate Cancer and it's usually account for 3% of all PaC in men at present (8). Although in most of the patients' disease have shown significant responses to this therapy but at later stages disease ultimately progresses known as Metastatic Castration Resistant Prostate Cancer (mCRPC). A highly metastatic with aggressive stage which is associated with consecutive rise in serum prostate specific antigen (PSA) levels or/and advancement of metastatic extent in the setting of castrate heights of testosterone and all treatment modalities are shown in figure 03 (9).
Figure 03: First step to treat disease is active surveillance. Current treatment options involves surgical resection at tumor site (prostatectomy), radiation therapy, cryotherapy therapy and/or use of systemic therapy including chemotherapy, hormonal therapy (luteinizing hormone releasing hormone antagonist: LHRH, androgen depletion therapy: ADT, pregnenolone antagonist: PA) immunotherapy (adoptive transfer of ex vivo activated T cells, natural killer cells, oncolytic viruses, cancer vaccines and administration of antibodies or recombinant proteins (3).

Genetic players in pathogenesis of PaC:
In approximately 5-10% cases PaC is inherited, which reflects a high percentage. The PaC is more aggressive in men with certain types of genetic mutations as compared to those without these mutations. Factors suggesting involvement of a genetic factor in PaC include: 1-Family history of multiple affected first degree relatives having PaC, especially if it runs in maternal or paternal family for three successive generations. 2-Early age onset of PaC (≤ 55y) as shown in figure 01. 3-People with family history of other cancers like breast and ovarian cancer have increased risk of developing PaC. As some genes involved in PaC are also associated with onset of ovarian and breast cancer. So there is 50% chance of passing on these genes to the off springs (10). Genetic defects associated with onset of PaC take into effect because of lower expression of tumor suppressor genes, over expression of oncogenes and oncofetal genes and due to mutations in many other cell cycle regulatory genes/ transcription factors and promoter sites.

PaC initiation and progression:
The initiation of PaC till aggressive stage development involves a series of genetic mutations in a distinct order. NKX3.1 haplo in sufficiency is the starting event in tumorigenesis of prostate, as has been proved by multiple Nkx3.1 knockout GEMMs. This mutation at chromosome 8p21 lead to consequences such as loss of basal cells, loss of markers of secretory differentiation and increase in potential to proliferate leading to prostatic intraepithelial neoplasia (early invasive cancer). Following this, deletion mutation in PTEN gene at chromosome 19q23 and homozygous deletion mutation in Rb gene at chromosome13q14.2 results in loss of basal lamina (invasive cancer). A SNP at chromosome 17p13.1 in p53 leads to onset of mCRPC (11).
Furthermore, in the case of mCRPC, recurrent aberrations involving the AR, ERG, Tp53, RB1, SPOP, CHD1, ZBTB16/PLZF and sections of chromosome 8q gain (including the MYC locus) and 8p loss are also observed. The researcher with coworkers also stated frequent mutations in FOXA2, which translates a transcription factor expressed in tumors exhibiting neuroendocrine features i.e. metastases from 05 men exhibited hyper mutated genomes with complex structural aberrations in MSH2 and MSH6 mismatch repair genes. All metastases and the primary tumors from these men were hyper mutated, demonstrating that mismatch repair deficiency observed early in the beginning of these carcinomas as shown in figure 04 (12). The tables 01-03 below have shown the major genetic multiplayers that contribute to normal development, initiation of PaC and progression to metastatic stage.  Growth Factor EGF-TGFα is mitogenic polypeptide.
it is activated upon binding with receptors having protein kinase activity for cell signaling TGF-α is transforming growth factor, it behaves as a ligand for epidermal growth factor receptor. It triggers signaling pathways for cell development, proliferation and differentiation The alteration frequencies of several genes and pathways are higher in metastatic samples.

Hormonal implication and molecular events associated with PaC:
The androgen biosynthesis is mainly regulated by GnRH that is released by the pituitary gland and leads to the synthesis of testosterone and DHT which are androgen receptor (AR) agonists. In the case of CRPC the overstimulation of AR and a subsequent increase in PSA is observed as shown in figure 05 (40). Various therapies tend to decrease androgen production via the canonical pathway 5. However, various alternative routes tend to ensure that the concentration of endogenous AR agonist such as DHT doesn't decrease thereby decreasing the efficacy of ADT. Moreover, undesired mutations can seriously augment the rate of biosynthesis and subsequently the tumor genesis of CRPC reported by Dai with coworkers in 2017 stated that by giving it a unique position within the steroidogenic pathway, and probably 3b-HSD1 was a critical enzymatic gatekeeper that deliberates on tumors the ability to connect adrenal androgens (40). In fact, a gain-of-function missense in 3bHSD1 has recently been described, which remarkably augments the capacity of this enzyme to drive conversion of DHEA to AD, thereby permitting more efficient DHT synthesis. This missense arises from a SNP at position 1245 (A to C), replacing an asparagine for threonine at amino acid position 367. The functional significance of this alteration, which can occur as either a somatic mutation or germline alternative, is an enzyme protein product that is concentrated resistant to ubiquitin mediated degradation, subsequent in intracellular accumulation. These include amplification and overexpression of AR, acquirement of constitutively active AR splice alternatives, deregulated AR coactivators/corepressors that sensitize AR in reaction to ligand binding, intracrine androgen synthesis, gain-of-function mutations and ligand-independent signaling and redundant downstream crosstalk.
Alongside aberrations in the enzymes of the biosynthesis pathways of androgens various other deleterious modifications have been observed within the Androgen/AR axis such as androgen receptor mutations/amplification loss of AR corepressor NCOR1/2 and gain of AR coactivator NCOA1/2 which leads to castration resistance. Also, conformational changes within the AR have also been noted due to genome structural changes within one third of the male population that suffers from CRPC. The abnormal expression of wide variety of AR variant species deficient in ligand-binding domain lead to persistent activation of AR signaling, such as AR variant 7 is suspected to steer disease progression (41).
Metastasis is also one of the leading pathologies associated with CRPC where the bones and lymph nodes are to be the most acutely targeted tissues. The damage to these organs and tissues leads to prevailing co-morbidities such as leukemia as blood cells fail to undergo proper specialization with decreased immunity. Recently, it has been discovered that TNF-α has been implicated in the progression of metastasis in all those individuals that suffer from CRPC and initiates the CCR7 upregulation that leads to cell migration of tumors and cause lymphatic metastasis (42). First, human PaC was observed to express both CCR7 and TNF-α. Second, through phosphorylation of ERK, low concentrations of TNF-α was seen to induce CCR7 in PaC cells. Finally, the migration of cancerous cells through phosphorylation of the protein kinase p38 was seen to be promoted by CCL21. So, the results of Maolake and colleagues in 2018 suggested that TNF-a drive the induction of CCR7 expression and CCR7/CCL21/axis might escalate the potential of PaC to metastasize in lymph nodes (42).
Despite the fact that the mechanisms of PaC and its subsequent metastatic predispositions are not known a strong effort is underway in developing effective treatments against the disease. Both conventional and novel therapeutic strategies are being employed to cure the disease. The conventional options improve the prognosis of PaC but along with desired therapeutic effect, the side-effects are often severe to tolerate for a patient. Also, some cancers are resistant to chemo and radiotherapy or are inoperable, underlining the requirement to devise new therapeutic procedures. Oncolytic virotherapy is proving to be a promising treatment for treating cancers with least side effects in recent era (7). The idea of activating patient's immune system against cancer-cells dates back. Immuno-oncology is an uprising field of cancer treatment with the ability to manage numerous malignancies with major goal of suppression of anti-cancer immune responses of body (43). Furthermore, novel therapeutics are also under developed where gene-based modalities gaining traction in recent years.

CHEMOTHERAPY FOR PROSTATE CANCER:
Under the scope of conventional therapy comes chemotherapeutic agents which are mainly small molecule drugs. Small Molecule Drugs are chemically synthesized and have molecular weight of <900da, cheap, readily available and administered orally. Furthermore, these agents are used in placebo and in combination in order gain maximum efficacy. The impact of chemotherapy on survival of PaC patients was first determined in CRPC in 2004 and since then further studies have demarcated the role of chemotherapy in increasingly earlier scenarios in disease presentation (44).
Cyclophosphamide: It is an alkylating agent that was used as part of clinical trial study against PaC. Researchers created three branches of the study Castration Resistant (CR), Progression Resistant (PR) and Stable disease. Little to no effect was seen in the first two branches with most of the patients in the study falling under the stable category. No effect was seen using the agent but interest was relighted when the drug showed potential for angiogenesis inhibition (45).
Cisplatin is a platinum-containing compound that inhibits DNA synthesis by cross-linking and denaturing DNA strands. In 209 cases reviewed, cisplatin showed a modest antitumor activity with a PR in 12% (95% confidential interval [CI]: 4-20% and thus was continued to be investigated as a single agent and in combination.
Carboplatin is a cisplatin derivative that results in intra-and inter-strand cross-linkage DNA damage, had been studied in earlier trials as a single agent with minimal responses. However, when combined with other chemotherapy drugs such as Paclitaxel and Estramustine, declines in serum PSA levels have been observed.
Satraplatin, the first oral 4th generation platinum analog found to be effective against cisplatinand carboplatin-resistant cell lines held a lot of promise in castration-resistant PaC and while it showed improvement in time to pain progression, it failed to improve overall survival (OS) in the Phase III SPARC registration trials (45).
Doxorubicin is an anthracycline that interdigitate between DNA base pairs and impairs topoisomerase II function inhibiting replication and transcription. Doxorubicin has also shown only marginal responses against PaC. In the earliest trials, responses varied from a 29% response rate as reported by The NPCP results showed clinical benefit with a response rate that included stable disease reaching 84%. Subsequent trials utilizing additional Ketoconazole with Doxorubicin alternating with Vinblastine and etoposide chemotherapy showed no additional benefit to hormonal therapy alone (45).
Eight therapeutic agents have been approved from the Food and Drug Administration since 2004 for management of patients with advanced PaC. There are four androgen signaling targeted inhibitors that derail androgen-receptor (AR) function (Enzalutamide, Abiraterone, Darolutamide and Apalutamide), 1 is an autologous cell based immunotherapy, 2 are Taxane chemotherapies that repress microtubule dynamics (Cabazitaxel and Docetaxel), 1 is a bone-targeted α-emitting radiopharmaceutical agent (radium223) and vaccine for PaC (Sipuleucel-T) (46). Docetaxel is a taxane derivative that works by binding to microtubules and preventing androgen receptor nuclear translocation and causing apoptosis through B-cell lymphoma (Bcl-2) phosphorylation (45). It inhibits microtubule disassembly and has demonstrated to downregulate AR transcriptional activity by inhibiting the translocation of the AR to the nucleus in response to both androgens and ligand-dependent signaling path-ways and known as an antimitotic agent. It also inhibits AR gene expression by acting on the gene promoter and rises the levels of Forkhead box O1 (FOXO1), a strong transcriptional repressor of AR (47). In addition, docetaxel also has anti-B-Cell lymphoma (BCL)-2 and anti-BCLX properties, thus promoting the self-eating by cell. Resistance to docetaxel may develop through several mechanisms including drug efflux, alterations in microtubule structure, activation of survival pathways, and changes in the tumor vasculature. In an effort to overcome resistance, investigators looked at combining targeted therapies with Docetaxel (48).
Cabazitaxel is a third-generation, semisynthetic tubulin-binding taxane drug and found to be as potent as docetaxel in cell lines and has antitumor activity in models resistant to paclitaxel and docetaxel (45). It prevents micro-tubule disassembly and shows antineoplastic action in cell lines with p-glycoprotein overexpression, which is also an anticipated mechanism for development of Docetaxel resistance. Moreover, Cabazitaxel has ability to penetrate the blood-brain barrier in preclinical systems and also hinder nuclear AR transport (49). It therefore, remains an option for patients with mCRPC who have failed with docetaxel. The potential advantages are: (1) the diminished affinity for P-glycoprotein, an important drug efflux pump and (2) the ability to cross the blood-brain-barrier (48).

Adverse effects of Chemotherapy:
Chemo-drugs for PaC are commonly administer through IV route while some drugs, such as Estramustine, are given through enteral route. The duration of therapy for advanced PaC depends on how well it is working and what side effects are appearing in patient's body. Possible Side effects may include: headache, fatigue, memory impairment, numbness, weakness, diarrhea, hair loss, mouth sores, emesis, anorexia and increased chances of infections (48). Some clinical manifestations are mostly specific with certain chemo drugs. For example: docetaxel associated with alopecia and bone marrow suppression but cabazitaxel causes neutropenia, anemia, thrombocytopenia, constipation, and lethargy. Estramustine have an increased risk of blood clots. Mitoxantrone rarely cause leukemia but it occurs several years later (45).

Mechanisms of Resistance to Chemotherapy
The resistance to chemotherapeutic agent after use attributes to particular mechanisms fundamental to PaC biology or broad-spectrum mechanisms as shown in table 04 given below. Anti-apoptosis Anti-apoptosis by inhibition of BCL2, BCLX, and clusterin and upregulation of prosurvival cellular pathways such as PI3K, mTOR, PKB, and angiogenesis by HIF, VEGF, FGF, NF-κB

Ineffective drug delivery
Ineffective drug delivery because of lack of lymphatic vessels and spherule formation by cancer cells

Paracrine cytokine secretion
Paracrine cytokine secretion induced by chemotherapy alters bone microenvironment, leading to tumor proliferation.

Up-regulation of pglycoprotein
Up-regulation of p-glycoprotein encoded by MDR1 gene and ABCB1 encoded by MDR2 gene leads to drug efflux.

Microtubule alterations
Microtubule alterations mediated by β-tubulin mutations leading to antiapoptosis as well as chemotherapy resistance.

Upcoming Therapeutic Targets:
Despite significant advances in the treatment of PaC using chemotherapeutic agents, the cure for the disease still remains a paradox. Alongside a lack of understanding of the already known mechanisms of the disease, new pathophysiological pathways are being discovered that further add to the complexity of the disease. Challenging as they may seem, these new pathways can also serve as potential avenues for a possible cure. Poly(adenosine diphosphate-ribose) polymerase (PARP) is associated with DNA repair and latest studies have confirmed an 11.8% occurrence of germline mutations in DNA repair genes in PaC. Inhibition of PARP has verified anticancer activity. In a phase 2 trial (TOPARP), the PARP inhibitor Olaparib confirmed better results in patients with mCRPC (50). Furthermore, Maoloake et al., 2018 also state that the CCR7/CCL21 axis can serve as a "Novel" biochemical pathway that can be targeted for the inhibition of the severe Metastatic potential of mCRPC if not the cancer itself (42).
The main purpose of the chemotherapeutic drugs is to rapidly kill or slow down the proliferation rate of rapidly dividing cancer cells. However, there are also non-cancerous cells in the GIT, bone marrow and hair follicles those divide rapidly. These cells are also destroyed due to the administration of the chemotherapeutic agents that lead to adversarial effects such as cognitive deficits including change in concentration, mental speed and memory, bone density reduction and fatigue (51). PaC still a leading cause of death in elderly men worldwide despite of advances in chemotherapy, radiation and surgery. Therefore, it is advisable for the scientific community to explore more diverse and effective options for PaC treatment.
Chimeric antigen receptor (CAR) T-cell therapy is a promising new way to get immune cells called T cells to fight cancer by changing them in the lab so they can find and destroy cancer cells Examples of CAR T-cell therapies currently approved include: Tisagenlecleucel (Kymriah), Axicabtagene ciloleucel (Yescarta), Brexucabtagene autoleucel (Tecartus) and Lisocabtagene maraleucel (Breyanzi). They cause cytokine release syndrome (CRS). Other serious side effects include neurotoxicity or changes in the brain that cause swelling, confusion, seizures, or severe headaches (54).
Immunomodulators target the immune system directly by turning down some proteins and turning up others. Thalidomide (Thalomid), lenalidomide (Revlimid) and pomalidomide (Pomalyst) are known as immunomodulating drugs (or IMiDs). These drugs can cause low blood cell counts, and neuropathy. PD-1 is a checkpoint protein, are given by IV (intravenously). Examples of drugs that target PD-1 includes the Pembrolizumab (Keytruda), Nivolumab (Opdivo) and Cemiplimab (Libtayo) and have been shown to be helpful in treating several types of cancer including prostate (Kaunitz et al., 2017). PD-L1 inhibitors includes the Atezolizumab (Tecentriq), Avelumab (Bavencio) and Durvalumab (Imfinzi). CTLA-4 is another protein to keep the immune system in check. Ipilimumab is a monoclonal antibody that attaches to CTLA-4 and stops it from working (55).
Sipuleucel-T (Provenge) is vaccines used to treat advanced PaC that is no longer being helped by hormone therapy (55). Interleukins are a group of cytokines that act as chemical signals between white blood cells such as IL-7, IL-12, and IL-21, continue to be studied against prostate cancer, both as adjuvants and as stand-alone agents (Bayer et al., 2019). IFN-α boosts the ability of certain immune cells to attack PaC cells. It may also slow the growth of cancer cells directly, as well as the blood vessels that tumors need to grow (54).
PVSRIPO stands for recombinant nonpathogenic oncolytic polio-rhinovirus chimera. This genetically modified agent was generated in part by replacing an intrinsic genetic component critical for viral protein translation (IRES element) with an equivalent component of human rhinovirus type 2. This change prohibited viral replication in normal neuronal cells, but supported replication in tumors of the central nervous system and elsewhere (51).
Some of the benefits of Oncolytic virotherapy is less invasive and debilitating; highly selective. Injections can be administered locally and uptake by tumor cells is increased. The viral machinery is manipulated to select and replicate within tumor cells only which limits the probability of infection in non-cancer cells. The large RNA size of Poliovirus makes it a favorable candidate to undergo genetic manipulations (56). Some of the risks as certain viruses and tumors require different dosage paradigms there is no standard dose for virotherapy currently. The optimization of viral production will be difficult since little information about cost is available. The administration, transport, storage and disposal of these viral agents will be a challenge for hospitals. Although, these viruses are weakened or attenuated, they still pose a threat to immunocompromised patients (57).
PVSRIPO may have advantage in dividing in cancer cells owing to broad phenomenon of mitogenic signaling cascade deregulation favoring cap independent translation. For example, the activation of MAPK-interacting kinase has a downstream effect of repressing serine-arginine-rich protein kinase (SRPK), which plays pivotal role in cap-dependent translation by acting on ITAFs (IRES trans-acting factor). The specific mechanism by which SRPK acts on viral translation in not known. PVSRIPO has proved potent oncolytic activity in a broad variety of cancer cell lines derived from gliomas, glioblastoma multiform, breast cancers, melanomas, Prostate cancer and astrocytoma (7).

Recommendations:
Investigators must focus on unraveling the complete biochemistry and pathophysiology of the disease before conclusively deciding the treatment modality. There is need for greater understanding of immune modulators, oncolytic viruses and phenotypic mixing. In phenotypic mixing, the surface protein of virus B can be coated on genome of virus A. This produced chimeric virus will now infect cells based upon surface recognition properties of B virus protein coat. However, the successive progeny of virus from this infection possess a type A coat as it is encoded exclusively by its type A genetic material. A fascinating instance of phenotypic mixing is that of pseudo-types, which consist of the nucleo-capsid of one virus and the envelope of another. The nucleo-capsid of vesicular stomatitis virus (a rhabdovirus) and the envelope of human immunodeficiency virus (HIV; a retrovirus) are currently being used to study the immune response to HIV17. Similar "Pseudo-types" can be developed and assigned specificity to target mCRPC cell lines and check fighting against disease.

Conclusion:
The latest proven prospective of oncolytic virotherapy e.g. PVSRIPO prove that persistent preclinical, mechanistic investigation solving all perspectives of the virus: host relationship have generated promising quantifiable outcomes in the most complex oncologic circumstances that has already become resilient to all available conventional rehabilitation approaches. However, it is important to note that the current progress of the experimental and clinical phases of PVSRIPO therapy, the possibility exists that this recombinant virus will soon be a viable treatment option for leukemia and solid neoplasia. Patient education in factors such as rate of success, cost, pre and post treatment procedures and health risks is of supreme importance before undergoing such types of treatment.

Compliance with Ethical Standards:
Not applicable