Therapeutic Value of Quinazoline-based Compounds in Prostate Cancer

Quinazoline Anti-Tumor Action

The anti-tumor activity of quinazoline-derived compounds has recently elicited exciting controversies in the field of prostate cancer therapeutics. The drugs were originally established to be efficacious in the treatment of BPH due to their ability to block the α1-adrenoreceptors, thereby reducing prostatic smooth muscle tone and relieving overall obstruction (23). Growing evidence from retrospective studies of BPH patients demonstrated that treatment with doxazosin and terazosin increased epithelial cell apoptosis in prostate tissue with no overall effect on the rate of cell proliferation (17, 18). Subsequent in vitro research revealed that the quinazolines' efficacy in inducing apoptosis was not limited to benign prostate epithelial cells but was also applicable to both androgen-dependent and CRPC cells. This effect was specific to quinazolines, as the sulfonamide-based α1-blocker tamsulosin failed to exhibit apoptotic activity (16, 19, 24). The molecular mechanisms via which the quinazoline compounds exert their pro-apoptotic effects are currently under intense investigation. Pre-clinical evidence suggests doxazosin mediates apoptosis in prostate epithelial cancer cells through the TGF-β signaling pathway, which controls cellular proliferation, differentiation, and apoptosis in numerous cell types. Doxazosin-activated transcription of the NF-κB inhibitor IκBα is another potential mechanism for the drug's increased apoptotic effects in CRPC, which exhibits constitutively activated NF-κB signaling (25, 26). Furthermore, more recent studies indicate that doxazosin induces apoptosis via the FADD-mediated Fas death receptor signaling pathway (27, 28).

Initial evidence of the quinazoline compounds' anti-angiogenic activity arose in a retrospective study of prostate cancer specimens in which tumors from patients treated with terazosin showed significantly reduced microvessel density compared to untreated specimens (13). Additional evidence revealed that terazosin inhibited VEGF-induced angiogenesis in both cultured HUVEC cells and an in vivo nude mouse model (14). Furthermore, doxazosin significantly impairs human endothelial cells by blocking cell adhesion, migration, and invasion due to loss of appropriate interaction between epithelial and endothelial cells and the extracellular matrix (ECM) (15).

Cell-ECM Interactions in Cancer Progression

The ability of endothelial and tumor cells to survive in the absence of proper ECM interactions is one of the driving forces behind tumor angiogenesis and metastasis. Cell attachment to the ECM is primarily mediated by integrins, which interact with fibronectin, laminin and a variety of other ECM proteins. Ligated integrins promote the formation of focal adhesion complexes, cytoskeleton-anchored signaling complexes associated with a number of non-receptor tyrosine kinases, such as focal adhesion kinases (FAK) and Src; actin-binding proteins, including talin and paxillin; as well as several other intracellular signaling molecules (27, 29, 30). Normal epithelial and endothelial cells undergo anoikis upon loss of integrin-mediated adhesion to the ECM, but malignantly-transformed cells can survive in an unanchored state by deregulating integrin-mediated survival pathways (31).

De-regulation of integrin signaling has been implicated in several types of cancer, including prostate cancer. FAK is expressed at very low levels in normal prostate secretory epithelium, while significant expression is observed at the earliest stages of transformation and continues throughout cancer progression (32, 33). FAK interacts with Src, which is also highly expressed in prostate cancer specimens and influences a variety of signaling pathways that affect cell survival, angiogenesis, proliferation, and motility (34). Talin-1 is significantly up-regulated in prostate tumor cells, leading to downstream activation of FAK/AKT signaling and, ultimately, anoikis resistance (31, 35). In a recent study, knockdown of β1-integrins in PC3-mm² cells abrogated anoikis resistance, and treatment with the β1-integrin-neutralizing antibody mAB 33B6 reduced tumor metastases in vivo, further implicating deregulated integrin signaling in the anchorage-free survival of malignant cells (36).

Reversal of anoikis resistance via targeting of integrin-mediated survival pathways is one of the most promising strategies for combating tumor metastasis and angiogenesis, and quinazoline-based compounds have demonstrated such activity in multiple cancer cell lines (27, 37, 38). Doxazosin reduces FAK expression in PC-3 cells and also exerts a modest disruptive effect on ILK-1, FAK, and paxillin binding with integrin-β1 in the human renal cell carcinoma (RCC) line 786-0 (27, 37). The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib reduces cell adhesion ability to fibronectin; reduces expression of integrin-α3, αv, β1, β4, β5, β6; and suppresses FAK phosphorylation in the oral squamous cell carcinoma line GFP-SAS-L1 (38).

Pharmacological Exploitation

The anti-tumor activity of quinazoline-based compounds has generated interest in the development of more potent novel compounds that maintain the quinazoline functional group (16, 24). In 2009, Giardinà et al. demonstrated that a related novel compound, cyclodoxazosin, exhibited greater anti-proliferative activity and repression of tumor vascularity compared to other novel quinazoline-based compounds. Cyclodoxazosin induced greater expression of caspase-3/7 and demonstrated greater growth inhibition of PC-3 cell line tumors inoculated in mice than doxazosin (22). In another study, the VEGFR2 inhibitor SKLB1002 significantly suppressed VEGF-induced phosphorylation of VEGFR2, extracellular signal-regulated kinases (ERK), FAK, and Src in endothelial cells. It also antagonized tumor angiogenesis in murine and zebrafish models without exerting any of the adverse side-effects associated with other VEGF receptor inhibitors, implying a therapeutic promise for optimized treatment modalities (39).

The two lead doxazosin-derived compounds developed and characterized by our group exhibited higher anti-vascular and anti-tumor activity than their parent compound, both in vitro and in vivo (21). The novel agent DZ-3 induced classical apoptosis in prostatic cells, while the new compound DZ-50 induced cell death and targeted tumor vasculature via anoikis. DZ-50 significantly reduced the viability of multiple epithelial and endothelial cell lines, and PC-3 cells exposed to DZ-50 showed decreased cell adhesion to fibronectin and collagen. DZ-50 was effective in impairing prostate cancer cell invasion in vivo and more importantly, treatment with DZ-50 reduced primary tumor growth and formation of metastatic foci in an in vivo model system (21).

More recent studies from our laboratory established the anti-tumor effects of DZ-50 against human renal cancer. Using two human renal cancer cell (RCC) lines as experimental models, 786-0 and Caki, we documented that DZ-50 induced early apoptosis at lower doses compared to the parent compound doxazosin (27). Both drugs significantly decreased the adhesion potential of RCC to fibronectin and laminin in a time-dependent manner and suppressed the cells' migratory and invasive capabilities. Our results clearly demonstrated that in response to DZ-50, RCC metastasis was significantly impaired in vivo via anoikis induction. Mechanistically, reversal of anoikis resistance by DZ-50 proceeded via decreased phosphorylation of FAK, AKT, and GSK-β, thus inactivating critical cellular survival pathways. DZ-50 also exerted a more potent inhibitory effect than doxazosin on ILK-1, FAK, and paxilin binding to integrin-β1. Collectively these results support the concept that doxazosin induces apoptosis via caspase-8 cleavage and subsequent caspase-3 activation, while DZ-50 invokes anoikis susceptibility by disrupting integrin/FAK-mediated cell survival pathways (27).

Multiple pre-clinical studies have analyzed the anticancer effects of quinazoline-derived compounds in combination with cytotoxic agents and other treatment modalities (40, 41). In one such study, the anti-tumor effects of the VEGF receptor inhibitor cediranib alone and in combination with a number of other compounds, including gefitinib, bevacizumab, docetaxel, and cisplatin, were compared in various tumor xenograft models. In each case, combining cediranib with another anticancer agent led to greater activity than either agent alone, with some combinations producing near 100% growth inhibition or even tumor regression (40). Moreover, an independent group of investigators evaluated the effects of a combination of terazosin and the soy isoflavone genistein on the prostate cancer cell line DU-145 (41). Simultaneous treatment with the two compounds more effectively inhibited cell growth than either compound alone due to a significant increase in apoptosis. In comparison to mono-treatment, treatment with both compounds significantly decreased expression of procaspase-3, Bcl-XL, and VEGF mRNA, while markedly increased PARP cleavage (41).

Potential Clinical Value

The lack of effective clinical treatment for CRPC combined with the promising anti-tumor effects demonstrated by quinazoline compounds in pre-clinical studies has generated significant interest in the potential clinical value of these compounds. In 2003, following several positive clinical evaluations (42-44), gefitinib was FDA-approved as a monotherapy treatment for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) after failure of both platinum-based and docetaxel chemotherapies (45). Encouraging results from pre-clinical studies (46, 47) and a phase I trial of gefitinib in patients with advanced solid tumors, including prostate cancer (48), inspired a phase II study of two doses of gefitinib in patients with CRPC (49). However, this study failed to demonstrate any activity for either dosage of gefitinib as monotherapy in CRPC. In another phase II trial of prednisone with or without gefitinib in the treatment of CRPC, gefitinib demonstrated only very moderate activity (50).

The failure of gefitinib to elicit a survival benefit in CRPC patients is disappointing, but clinical trials of other quinazoline-derived compounds have produced more promising results (51, 52). For example, a recent phase II trial evaluated cediranib in patients with metastatic CRPC who had progressed after docetaxel therapy (52). Among patients with measurable disease, six had confirmed partial responses and one had an unconfirmed partial response. The probability of progression-free survival (PFS) for all evaluable patients (n=58) was 43.9% at 6 months, median PFS was 3.7 months, and median overall survival (OS) was 10.1 months. Vascular permeability, which was assessed by dynamic contrast-enhanced magnetic resonance imaging, was reduced across large proportions of patients, as evidenced by statistically significant reductions in gadolinium uptake. The most common toxicities were hypertension, fatigue, anorexia, and weight loss, and these effects were reduced by concurrent treatment with prednisone (52).

Recently gathered pre-clinical evidence suggests cooperative anti-tumor effects of quinazoline-based compounds in combination with other treatment modalities (40, 41). Clinical trials of such combination therapies, however, have produced controversial results (53, 54, 55). For example, a phase II trial of docetaxel with erlotinib in elderly patients with CRPC failed to demonstrate a survival benefit by treatment with both compounds (54). On the other hand, a phase II trial evaluating carboplatin and paclitaxel in combination with either cediranib or placebo in patients with advanced NSCLC produced more encouraging results (55). There was a significantly higher response rate for cediranib than for placebo, with a hazard ratio of 0.77 for PFS, and a similar number of deaths in both arms. However, cediranib patients experienced more adverse effects, including hypertension, hypothyroidism, hand-foot syndrome, and GI toxicity. Concerns over tolerability precluded immediate advancement to phase III testing (55), but these results still support further exploration of quinazoline-derived compounds in combination therapies.

The anti-tumor effects of quinazoline-derived compounds observed at the cellular level may also translate to a chemopreventative action. In 2007, we first reported the results from a retrospective study in which the medical records of Lexington, KY, VA Medical Center patients treated with doxazosin or terazosin were reviewed and compared with the Markey Cancer Center's Kentucky Cancer Registry, a state-based portion of the NCI's Surveillance, Epidemiology, and End Results (SEER) program. Patients treated with the α1-adrenoreceptor antagonists showed an overall prostate cancer incidence of 1.65% compared to 2.41% in untreated patients, which is equal to a 1.46-times lower relative risk and 31.7% lower attributable risk of prostate cancer development in treated vs. untreated patients (56). These results yielded much promise and generated momentum towards conducting an expanded prospective, multi-center trial comparing prostate cancer incidence among BPH patients treated with α1-adrenoreceptor agonists vs. those treated with placebo. Such a study could include an evaluation of the results of monotreatment with various quinazoline-based compounds, which could ultimately lead to the identification of a parent compound to be utilized in the optimization of new chemical structures with greater preventative capabilities.