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AP : FRIEND OR FOE? POTENTIAL ROLE OF ACID PHOSPHATASE IN CARCINOMA OF THE PROSTATE
ADITYA PAREEK
Department of Urology, Bombay Hospital, Mumbai, India.
Malignant potential of carcinoma of the prostate (CaP) is so variable that many patients die with the disease, from causes unrelated to it or simply old age, with or without concomitant progression of the tumour, where others develop rapidly progressive disease which is fatal within a short period. Biomolecular mechanisms of progression of the CaP remain unknown despite extensive research. Research shows that there occur alterations in the activity and/or response of various growth factors and their receptors, among other biomolecules, active in cell division. However, none of these changes are consistently observed to a similar extent in all tumours.
Immunohistochemistry consistently shows that the intracellular acid phosphatase (AP) contents of the prostate cancer cells are low as compared to that of normal prostate epithelial cells. Greater the tumoral de-differentiation, lesser is the immuno staining of AP. Serum AP levels remain low/normal in these types. These types of tumours show little, if any, response to hormonal treatment. Apart from these varieties, majority of the CaPs on progression show rising serum AP levels despite low intracellular AP contents. Raised/rising serum AP level in the cases with progressing tumours is usually explained by a high rate of cell attrition though this doesnot explain the concomitant low intracellular AP. This suggests that the AP synthesis per unit of cancer mass is depressed and/or progressing tumours maintain low intracellular AP by accelerated pumping of this enzyme outside the cells.
Certain drugs used in the treatment of advanced CaP are now suspected to accelerate the tumour progression. Chemical structure of these compounds shows that the radicals contained in these are known inhibitors to the AP activity. Since the AP has been shown to have protein tyrosyl-phosphatase (PTP) activity, probably involved in the regulating the action of stimulatory growth factors by controlling the protein-tyrosine-kinase (PTK) activity, it is possible that these drugs may accelerate CaP progression by tipping the balance in favour of PTK. It follows, therefore, that the low intracellular AP may be one of the mechanisms driving the cancer cell to perpetual cell division.
INTRODUCTION
Biological function of acid phosphatase (AP) remains unknown even though this enzyme has been extensively investigated and used as a tumour marker in patients with carcinoma of the prostate (CaP) for over half a century. The AP is therefore called a non-functioning enzyme.[1] It is widely distributed in a variety of tissues particularly the red blood cells, osteoclasts, kidney, liver and prostate; of all the tissues, prostate is the richest source of this enzyme. Because of various sources of AP, identification of the tissue of origin of the enzyme becomes important in any clinical or laboratory investigation. Several methods exist.[2] In enzymatic methods, differential inhibition of AP from different tissues by various substrates and inhibitors identifies various isoforms. Classical inhibitors of the prostatic AP are various organic phosphates, molybdate, vandate, L+tartrate and fluoride. [2,3,4] Most sensitive substrate of the prostatic AP is thymophthalein phosphate though this reaction is also not absolutely specific. [2,5] Immunoassays are based on recognition of specific binding sites on its molecule by specific antibody. Several isoforms of the enzyme can be further characterized. It appears that the isoform 2 and probably also 4 are relatively ‘more specific’. [5] Therefore, there is no method which can distinguish completely and absolutely the sources of serum AP. For this reason and for the fact that various isoforms are distinguished by its action on artificial substrates, the enzyme will be simply referred to as AP in this article.
In biological systems, the AP functions as phosphomonoesterase like alkaline phosphatase, shuttling phosphate radical in dephosphorylation / transphosphorylation reactions. [1,3,6] Its natural substrate in semen is phosphorylcholine though the physiological significance of this reaction is unknown. It may be noted that lysosomal AP is also present in prostates as in other tissues (except red blood cells). Lysosomal and secretory forms of the enzyme are encoded by different genes. [3]
AP and CaP
After synthesis probably in rough endoplasmic reticulum, the AP is stored in supranuclear vesicles as shown by intense immunostaining of apical regions of normal and benign prostate epithelial cells; this staining, like that of prostate specific antigen (PSA), becomes progressively less marked and patchy in well, moderately and poorly differentiated tumours.[7,8] This shows that as the CaP becomes less differentiated, the AP (and PSA) content per cell is progressively reduced. With malignant transformation, the PSA content of the cells decreases first, followed by that of AP, which is a reversal of the process seen in foetal prostate where AP appears first followed by PSA.[8] Serum AP levels begin rising in cases with tumours on progression which is alarming to clinician. Upward trend in PSA precedes tumour progression by several months.[9] Thus, the tumour markers are viewed as devil’s messengers. Do all these observations indicate that the cancer cell membrane is excessively leaky to these enzymes? However, the serum levels and tumoral intracellular (AP and PSA) content are very low in cases with anaplastic or neuro-endocrine de-differentiation.[8,10] This suggests that the tumour marker synthesis is depressed in these histological types. Whatever the mechanism(s), intracellular AP and PSA concentration of cancer cells is low as compared to that of normal/benign prostate epithelial cells. These facts coupled with certain experimental and clinical studies (see below) suggest that the AP may play a role in progression of CaP.
AP, CaP and Endocrine Treatment
Endocrine treatment of CaP is basically palliative. Response to first order endocrine treatment varies between 30%-80%.[11-16] Those which respond initially, majority of these relapse in about 18-36 months. [13] Response to second line endocrine treatment and chemotherapy is generally poor, and most of the patients with hormone refractory disease die within a few months. Various mechanisms of primary and secondary resistance to endocrine treatment have been postulated (see below).
Of several drugs, drug combinations, and orchidectomy, none has been found to be superior over another in terms of progression free or overall survival. [17] It is clear however that with hormone naive disease, a higher proportion of patients on MAB (Maximal Androgen Blockade) show tumour marker response as compared to that in those on LHRHa/orchidectomy monotherapy. [18-22] There are few studies on the effect of anti-androgens on hormone refractory disease because majority of these patients by this stage are in very poor general condition and most of them die within a few months. In these cases also, the tumour marker response is seen in a proportion, but the duration of follow up is so short, it is almost impossible to predict how many more of them would have had decreasing serum levels of AP and PSA, had they survived long enough. It may be noted that serum AP levels continue to fall in hormone responsive CaP with primary hormonal treatment for over several months. [22] Thus, despite tumour marker response in a proportion of the patients with hormone refractory disease who are alive long enough to be observed, no objective clinical response is seen either with anti-androgen monotherapy or MAB. [23-25] Previous studies with oestrogens have also shown that the reduction in serum AP levels may occur in upto 45% of the patients with hormone refractory disease without however, any objective clinical response. [26-27]
It is clear, therefore, that the tumour marker response (AP and PSA) is only indirectly related to any form of hormonal treatment, and cannot be taken as a surrogate end point of tumoral response either in previously hormone naive or hormone refractory disease. [17,28,29] From abovementioned studies, one fact emerges : the MAB regimen lowers tumour markers better than any form of monotherapy. Finally, in the initial flare phenomenon caused by LHRHa monotherapy the rise in serum AP levels doesn’t occur if an anti-androgen is also added or started prior to LHRHa. [30-31]
Why blockade of the effect of residual androgens (of adrenal origin) by the additional use of an anti-androgen (MAB regimen) does not provide better tumour control despite relatively significantly better tumour marker response is not clear. On the contrary, certain recent observations reveal potentially adverse effects of the anti-androgens in tumour control.
FLUTAMIDE/ANTI-ANDROGEN WITHDRAWAL SYNDROME
Theoretically, flutamide and other anti-androgens should provide additional benefit probably by effecting rapid or extra cancer cell kill in patients on MAB. Actually, if the anti-androgens were reliable and complete androgen receptor blockers, these should have been effective as monotherapy, which is not the case. Monotherapy with non-steroidal anti-androgens (NSAA) is not generally recommended. Cyproterone acetate (CPA), a steroidal anti-androgen, is structurally very different from NSAA. Monotherapy with CPA is also not widely accepted.
Flutamide withdrawal has been reported to benefit upto 40% of the patients showing clinical deterioration on MAB. [32-34] If the clonal selection or development of secondary resistance occurs with the mechanism widely believed (see below), withdrawal of flutamide may not be expected to revert the resistant cells to sensitive state. Continued LHRHa therapy in "flutamide withdrawal responders" may be beneficial for about a year; this may mean that the fraction of the cancer cells selected as resistant in this group of the patients are sensitive to LHRHa therapy. Therefore, it seems likely that the flutamide simply drives the cancer cells to progression which stops on its discontinuation in about 40% of the patients. One possible explanation is an agonist effect of flutamide on, possibly a mutated androgen receptor (AR). It may be noted that AR mutations are quite uncommon in primary CaP and the chemical structure of NSAA is very different from either testosterone (T) or DHT. Also if the AR mutations occurred in the group of the patients showing deterioration on MAB, why other 60% don’t benefit following withdrawal of flutamide from the MAB regimen?
It is now suspected that the anti-androgens and indeed even oestrogens may actually be driving the ‘hormonally resistant’ cells to progression.[35] Clinical studies show that the admission of NSAA for initial treatment resulted in hormone resistance. [37] Experimentally, it has been shown that the androgen ablation and oestrogen treatment result in p125 FAK and paxillin activation by protein-tyrosine phosphorylation. [38] This is probably a very interesting observation.
So far, the possible mechanisms of tumour progression and hormonal resistance which have drawn most attention, are mutations and amplifications of AR/gene [39,40] loss/abnormalities of tumour suppressor and dominant oncogenes including DNA hypermethylation, [41,42] altered response and sensitivity of growth factors [41,43] and the clonal selection of primarily resistant cells by any of the mechanisms. [44] Since many of the abnormalities of DNA and growth factors appear to be common in varying proportions in many other cancers of various tissues, it is unlikely that the currently known molecular aberrations represent specific events for a given cancer. Given that the role of androgens in cell cycle control is permissive, [3,41] further research in the steps of afferent and efferent loop(s) to and from genome may unravel the complexities of hormonal resistance of CaP. Research may also help explain the reasons of probable acceleration of tumour progression in some patients given anti-androgen therapy.
INTERACTION OF AP AND GROWTH FACTORS WITH ENDOCRINE TREATMENT
Effector arm of the androgens in cell cycle control is probably a variety of growth factors.[3,41,43] Stimulatory growth factors, the EGF/TGFa and FGF-7 act via protein tyrosine kinase (PTK) activity of their receptors, driving IP3/DAG system. Inhibitory TGFb have serine/threonine kinase (STK) activity. It may be noted that the relevant protein phosphorylation effected by the given kinase must normally be balanced by dephosphorylation catalysed by a phosphatase resulting in signal abrogation at an appropriate stage. The phosphatases are not constitutively active and there may exist a regulatory control between kinases and phosphatases at appropriate stages of growth factor signals. [1,45]
It has been shown that the prostatic AP is the same enzyme as protein-tyrosyl-phosphatase (PTP). [4,46] This finding may have profound implications as discussed below.
It may be significant that the currently available NSAA contain fluoride ion. The fluoride ion is well known inhibitor of the activity of prostatic AP. Bicalutamide which contains four fluoride ions instead of three in flutamide and nilutamide, lowers serum prostatic AP in a dose dependent manner. [47] Flutamide and nilutamide may also be expected to act similarly. Oestrogens (e.g. Estmustine, stilboesterol) often used in the treatment of CaP contain phosphate group. In the process of oestrogen phosphate hydrolysis, some of the AP may be inactivated depending upon the concentration of oestrogen phosphate. Cyproterone and almost all LHRHa contain acetate. It is not clear from literature if the acetate coupled to these drugs can affect AP activity. Abnormal metabolism of NSAA and oestrogens (e.g. slow turnover) or abnormal response of AP/PTP to these drugs is another possibility.
Assuming that the inhibition of AP/PTP by fluoride ion (of NSAA) allows PTK activity of the relevant stimulatory receptors (EGFR) remaining unchecked, normal/low levels of ligands may continue to stimulate cell replication. In fact, the activity of TGFa, a stimulatory ligand for EGFRs, is actually increased in CaP cells; poorer the differentiation, more its immunoreactivity, [48] which appears inversally related to the activity of intracellular AP. Androgen withdrawal and oestrogen treatment induced p125 FAK and paxillion activation via tyrosine phosphorylation may impact increased motility and invasiveness to cancer cells.38 The possibility of a role of AP/PTP in regulation of this process may need investigation. The AP/PTP activity doesn’t appear to regulare STK activity. [4,46]
Homozygous deletion of the gene encoding "haematopoietic cell phosphatase" (hcp) leads to lethal overproliferation of a variety haematopoietic cells in "motheaten" mice. [49] Normally, down regulation of cytokine mediated signal transduction is brought about by the phosphatase causing dephosphorylation of receptor associated JAKs. [50] It follows therefore that a specific deficiency of SH-PTP 1 may be the cause of haematopoietic cell overproliferation. This serves to indicate that the PTP may play a regulatory role in cell replication and that the stereo-structure and docking sites of various PTP on target molecules may be tissue specific. In this context it may be interesting to note that leucoerythroblastic anaemia develops in about 45% of the patients with hormone refractory CaP. [51] Various SH-domains probably impart group specificity to PTPs in membrane binding of phospho-tyrosine in growth factor mediated PTK activation.
Focal heterogeneity and inactivation of MMAC 1/PTEN, a tumour suppressor gene located on 10q 23.3 has been reported in the CaP.[52,53] It is not clear whether the encoded protein phosphatase is actually the same enzyme as ‘acid’ phosphatase hypothesized to be involved in the given PTK regulation or some variant thereof. It is clear, however, that both (if different) the enzymes are reduced in the cells of CaP.
High phosphomonoester : phosphodiester ratio observed in tumour cells indicates increased rate of membrane synthesis and probably rapid cell proliferation. [54] Since the phosphomonoesters and their precursor, the phosphorylcholine (among others), are natural substrates of the AP, a low intracellular concentration of this enzyme may be a mechanism providing increased supply of the compounds for membrane synthesis. It may also be noted that the oncogene PTK activity results in the production of tyrosine-phosphoesters. Therefore, the role of several tissue-specific and nonspecific species of AP in membrane synthesis may need investigation. As indicated earlier, group specificity may be due to the nature of SH-domains.
Retinoids and short chain fatty acids inhibit growth of CaP cell line by inducing alkaline phosphatase. [55] This may be a non-specific esterase effect on cellular phosphomonoesters, causing retardation of membrane synthesis. The AP acts similarly on these substrates. The PSA is also an esterase, in addition to being serine protease. However, the intracellular alkaline phosphatase concentration is unlikely to be significant, if at all, in CaP cells. Interestingly, the intracellular pH of cancer cells is either neutral or alkaline due to overactive Na/H antiport. [56] It has been postulated that mild alkalization of the cell may play a role in stimulating mitosis. [1] Since the optimum pH for AP is 4-6, its activity is certain to decline in alkaline millieu.
Chemical structure of the NSAA (especially that of the flutamide) is strikingly similar to that of Sanger’s reagent (1-fluoro, 2.4 - dinitrobenzene), of which active fluoride (Fl) radical reacts with the -NH2 groups of amino acids in proteins, releasing hydrogen fluoride (HFI). Parent molecule of the Sanger’s reagent remains tightly bound to the amino acid, and this bond is resistant to acid hydrolysis.1 Several aminoacids including tyrosine react with the Sanger’s reagent in this way. Similar reaction can be expected between Fl of NSAA and aminoacid. Irreversible binding with tyrosine may alter/affect the results of PTK activity. However, perhaps it is more important that the release Fl reacts with and inactivates the AP/PTP. Of course, the Fl may have other effects in vivo such as the formation of fluroacetate with inhibition of Kreb’s Cycle. CPA is also known to decrease intracellular AP, [57] though the mechanism is not clear. This may be a part of general depression in protein synthesis resulting from low T levels. PSA synthesis is androgen dependent since castration lowers serum PSA and replacement of androgens increases it. [58] It is believed that AP synthesis is not androgen dependent. However, both the AP and PSA being glycoproteins, are expected to fall with lower over all protein synthesis as T levels decline. Major difference between CPA and gonadal/adrenal steroids is the presence of highly reactive = OCOCH3 and CH2 groups in the cyclopentanoperhydrophenanthrene nucleus of the former. One -CH3 group each at C18 and C19, a Cl at C6 and double bonds between C4-5 and C6-7 are also additional features of CPA. Fate of these radicals in vivo is not clear, though their presence on the parent molecule must be responsible for AP blockade and inhibition of 17,20 desmolase. On the other hand, very unstable nature of = OCOCH3 and = CH2 bonds, coupled with the fact that CPA very closely resembles gonadal steroids in structure, may mean that the binding of this drug may actually stimulate an appropriately infidal AR. This has not been proven. However, if the = OCOCH3 and = CH2 are cleaved off the parent molecule in vivo, what remains of CPA is basically a progesterone.
Mutations in AR gene have been described (e.g. codon 868: Thr/Ala), though these are very uncommon in primary tumours. It has been suggested that such and other changes in the hormone responsive domain of the AR may result in its being stimulated by not only the DHT which is a normal ligand, but also by oestrogens, flutamide, CPA and RU23908 (see review by Kelly and Slovin). [59] This implies that at least the part of the binding site on AR is common to all these agents. Since the concept of AR blockade is based on the similarity of over all structure between the natural ligand and the simulator drugs, and considering the expected limited number of chemical bindings the antiandrogens can therefore undergo, it would be surprising if these drugs always coupled with the variety of changed amino acid sequences described in AR mutations. And if such a coupling does occur, it is also surprising that this always stimulates the AR. Also, if the paradoxic response of AR to these drugs were due to AR mutations only, these specific altered amino acid sequences must be present in all Caps showing proliferative response to these drugs.
It appears that although the AR mutations are important in some of these patients, in others, alternative mechanisms must be sought, perhaps in the effector arm of the stimulus to tissue growth. Even in those patients with paradoxically responsive and mutated AR, it might still be possible to check the cell growth and replication by modulating the effector arm of the AR mediated response at growth factor/PTK level. It is clear that all antiandrogens reduce intracellular AP, the degree of which may depend on the nature and intracellular concentration of the drug, basal AP values and possibly on the variations in the stereo-structure of or the docking sites on the AP in different patient groups. Any beneficial effect of the drugs may be outbalanced in some patients by disproportionately marked inhibition of the AP. So although the antiadrogens block the normal AR, they at the same time activate the growth factor related mechanism by inhibiting the AP/PTP. And since this mechanism lies beyond the AR generated stimulus and now independent of it. The proliferative response ensues and sustains. Since the AP is known to have PTP activity, inhibition of intracellular AP may be common mechanism driving PTK unchecked, and therefore the cell replication, in a subset of the patients with CaP. Actually, there is growing body of evidence suggesting the regulatory role of PTP as a widespread mechanism.[45,49-50,60-61]
If the AP/PTP inhibition by NSAA and CPA were mechanism of unchecked cancer cell proliferation, why about 60% of the patients do not benefit following withdrawal of these drugs from MAB? It is unlikely though that the AP/PTP inhibition alone can explain hormonal resistance or tumoral progression in this group of the patients. However, it may be recalled that the intracellular AP content is roughly directly proportional to tumoral differentiation accepting however, the heterogeneity of CaP. Apart from possible diminished AP synthesis per unit cancer mass, the cancer cell membrane may be excessively permeable to AP. Whatever the mechanism(s) the intracellular AP concentration of CaP cells is low. This, the flutamide withdrawal non-responders may be the group whose CaP cells contain much less AP already, which means that these cancer cells may already have high unchecked PTK activity and are of poorer differentiation. As a corollary, flutamide withdrawal would benefit only those cases whose CaP cells have relatively high AP values than the previous group. This means that the tumours of flutamide withdrawal responders may be better differentiated. In well differentiated tumours with ‘nearly normal’ intracellular AP values the effects of NSAA may be minimal. It is also possible that variation in AP/PTP structure exists in different patient groups. It therefore appears that the prostate-cancer cells have developed a self sustaining replicatory process by maintaining the intracellular AP concentration low. In other words, this mechanism develops concomitantly with Carcinogenesis and provides the Cancer Cells the ability to proliferate freely. The mechanism of low AP concentration may have its origin at genome or in post-genomic collateral pathways. As indicated earlier, increased cell membrane permeability to AP is another or additional possibility.
Possibility of CaP progression with oestrogen therapy, due to AP/PTP inhibition by oestrogen coupled phosphate, has been explained above.
This hypothesis is not intended to explain the mechanism of hormonal resistance in CaP. It may explain the mechanism of tumour progression with anti-androgen therapy. Anti-androgen withdrawal from MAB regimen may allow restoration of AP/PTP activity to previous value which would provide braking effect on PTK. Possible beneficial effects of intermittent androgen suppression with MAB can also be explained by this mechanism. This may be the biological function of AP. Thus, the AP may be a friend and need not be inhibited.
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