Targeting EGFR with photodynamic therapy in combination with Erbitux enhances in vivo bladder tumor response

Photodynamic therapy (PDT) is a treatment modality that involves the administration of a tumor-localizing photosensitizer followed by light irradiation of specific wavelength that matches the absorption characteristics of the photosensitizer, thereby producing cytotoxic intermediates that damage cellular structures [1]. The advantages of PDT include selective targeting, minimal invasiveness and reduced toxicity that allows for repeated treatment [2, 3]. However during PDT, tumor oxygen is depleted due to vascular damage and oxygen consumption, which causes hypoxia within the surviving tumor cells thus triggering angiogenesis [4, 5]. Angiogenesis is the sprouting of new smaller vessels from the pre-existing vasculature. Not only is angiogenesis essential for tumor growth but it also enables the migration of tumor cells to distant sites, forming metastases [6].

Bladder cancer is the 9^th most common cancer affecting Singapore men [7]. Current treatment options include surgery, chemotherapy or immunotherapy, and radiation therapy [8]. Efforts are on going to develop therapeutic tools that allow the preservation of bladder and to control the rate of recurrences. Clinical trials with PDT have shown promising results in the treatment of bladder cancer, especially for flat malignant lesions such as carcinoma in situ [9, 10]. Recently, significant progress has also been made to understand the molecular and genetic events underlying bladder cancer [11]. Epidermal growth factor receptor (EGFR) is one such molecular marker that has been widely reported in bladder carcinoma [12, 13]. Upregulated EGFR signaling is known to initiate a cascade of events leading to cell proliferation, migration, invasion [14] and blocking of apoptosis [15] that eventually leads to tumor progression. Many epithelial cancers have been found to overexpress EGFR, including head and neck, breast, colon, lung, prostate, kidney and bladder [16]. Studies show that antibodies that block the EGF binding site of EGFR inhibit tumor cell proliferation [17]. Therefore, blocking EGFR along with conventional cancer therapies could be an attractive anti-tumor strategy.

Erbitux (cetuximab), a chimeric human-murine monoclonal antibody, competitively binds to the accessible extracellular domain of EGFR and inhibits dimerisation and subsequently inhibits cell proliferation, tumor growth and metastasis [18]. In most studies, the use of Erbitux, as an anti-EGFR therapy in combination with chemotherapy and radiotherapy has demonstrated significant clinical efficacy, due to its good tolerability and non-overlapping toxicities [19]. Also, in vivo therapies with Erbitux and chemotherapy drugs resulted in a greater regression of bladder tumor growth compared with either agent alone [20]. In the present study we have evaluated the anti-tumor effect of Erbitux in combination with PDT on bladder carcinoma xenograft model. Our findings indicate that combining PDT and Erbitux significantly enhances the anti-tumor activity, by inhibiting EGFR expression, increasing apoptosis and by dephosphorylating essential EGFR tyrosine sites. These results may provide a rationale for evaluating the combination of PDT and Erbitux as a cancer treatment modality in a clinical setting.

PDT is being successfully used in clinics for the treatment of superficial lesions of both malignant and non-malignant diseases. However, treating solid tumors is still a challenge due to issues related to penetration of light, non-homogeneity and geometry of the tumors [21]. Triggering of angiogenesis is also dependent on different PDT parameters such as drug/light dosage and drug light interval. Previous studies have shown that sub-optimal PDT elicits increased angiogenesis [22, 23]. In our earlier study we have reported that high dose light PDT with high fluence rate induces the overexpression of VEGF compared to low dose light PDT [24]. We have also noticed that predominantly cellular targeting long drug light interval PDT can induce greater expression of angiogenic proteins compared to vascular targeting short drug light interval PDT [25]. Therefore, there is a need for continued investigation to enhance the anti-tumor efficacy of PDT for improved response and expanded use. In this study, we evaluate the use of EGFR inhibitor Erbitux in combination with PDT to improve the tumor responsiveness in a bladder tumor xenograft model.

Bladder cancer treatment remains a challenge though significant progress has been made in the prevention of disease progression and the improvement of patient survival rates. PDT has been successfully used to treat recurrent or drug resistant superficial bladder cancer. 5-aminolevulinic acid (ALA)-PDT has shown to be an effective treatment option for patients with superficial bladder cancer [26]. However, ALA-PDT can cause pain and would require some form of local anesthesia. Some investigators have concluded that in most clinical trials of bladder cancer, the PDT treatment was overly aggressive and resulted in long lasting and severe urinary complications [27]. Nseyo et al. [28] suggested multiple treatments at lower drug and light doses to reduce the incidence and severity of symptoms following PDT of superficial bladder cancer. Single session whole bladder PDT using diffusion medium for isotropic light distribution was beneficial for patients treated with TCC refractory to traditional intravesical therapy [29]. However, patients with extensive flat papillary lesions did not appear to respond well. As can be seen, PDT treatment of bladder cancers continues to present major challenges and novel therapeutical approaches need to be explored.

Erbitux was approved by the US Food and Drug Administration (FDA) for use in combination with irinotecan for the treatment of metastatic colorectal cancer and it is also being used for the treatment of metastatic squamous cell carcinoma of the head and neck (SCCHN) [30, 31]. Results of a large phase II study on irinotecan-refractory colorectal cancer patients have shown a significant response of 22.9% when Erbitux was combined with chemotherapy agent, irinotecan [32]. In another study, the response rate was significantly improved when Erbitux was combined with cisplatin in the first-line treatment of recurrent or metastatic SCCHN [33]. A randomized trial that compared radiotherapy plus Erbitux with radiotherapy alone in patients with stage III or IV non-metastatic SCCHN, demonstrated significantly longer locoregional control with radiotherapy plus Erbitux than with radiotherapy alone; moreover, progression-free survival were significantly longer and the overall response rate was significantly better with the combination therapy [34]. Recent results from a phase III randomised study demonstrated that the Erbitux given concomitantly with radiotherapy yields a significant clinical benefit over radiotherapy alone without any increase in radiotherapy-associated toxicity [35].

In our in vivo tumor regression study, we demonstrate that the combination therapy of Erbitux with PDT can improve the tumor response by attenuating the angiogenic process. A similar study conducted on a mouse model of human ovarian cancer in which C225 (Erbitux) was combined with PDT regimen produced synergistic reductions in mean tumor burden and significantly increased median survival [36]. In this study, PDT treated tumors did not exhibit significant tumor regression compared to combination therapy groups and this could be attributed to the high fluence rate that was administered during PDT. High fluence rate can deplete tumor oxygen to a large extent, thereby stimulating the production of stress induced survival molecules that reduce the effectiveness of PDT and affect tumor control [4]. More importantly, the administration of high light dose for this experiment was to test our hypothesis that combining PDT with Erbitux can improve tumor control and also to evaluate the effectiveness of Erbitux in reducing EGFR concentrations. Our investigations have indicated that Erbitux alone as monotherapy was not effective in controlling tumor growth. One of the possible reasons for this observation could be the fact that tumors overexpressing EGFR might not be sensitive to Erbitux. Although we would assume that tumors overexpressing EGFR would respond well to anti-EGFR therapy, studies have demonstrated that the level of EGFR expression does not have any impact on tumor response rates as a significant number of EGFR-positive tumors could be resistant to Erbitux [37, 38]. The group that received the combination therapy of PDT and Erbitux exhibited accelerated growth a week after PDT which could be due to an increase in the expression of angiogenic growth factors either due to hypoxia, induced by oxygen depletion during PDT light irradiation or incomplete treatment. Our earlier results have shown increased expression of angiogenic growth factor VEGF at 72 h post PDT [39]. In this study, the regular administration of Erbitux after PDT treatment could have blocked the EGFR pathway and reduced angiogenesis. Therefore, our data supports the hypothesis that combination therapy of PDT and Erbitux would be more effective in preventing angiogenesis compared to monotherapy alone.

To further substantiate our results we performed western blotting, immunohistochemistry and immunofluorescence to determine the EGFR levels in all the treatment groups. EGFR immunoreactivity was localized mainly in the cell membranes and to a lower extent in the cytoplasm. It has been well established that the core of solid tumors is hypoxic, and that hypoxic tumor environment is sufficient to trigger EGFR expression in tumors [40]. Previous studies have reported the downregulation of EGFR after PDT [41, 42]; in marked contrast our results demonstrated an increase in EGFR expression post hypericin-mediated PDT. This observation could be attributed to numerous reasons such as the light/drug dosage, the complexity of tumor microenvironment and the properties of the photosensitizer [4]. Combined antitumor activity of Erbitux with standard chemotherapy and radiotherapy is well documented in the treatment of different types of tumors and is reported to be more efficacious than individual monotherapies [19]. In this study, combination modality of PDT and Erbitux was effective in reducing the expression of EGFR and that could have lead to the regression of tumors in this group.

In the current study, we have also shown that PDT plus Erbitux increased apoptosis in the treated tumors compared to PDT only and inhibitor only monotherapies. Erbitux has been known to increase apoptosis in various tumor models by different mechanisms, including upregulation of pro-apoptotic Bax protein [43], decrease in the expression of anti-apoptotic molecule Bcl-2 [44] and the activation of pro-apoptotic caspases [45]. Hypericin-PDT is also known to induce apoptosis in a dose-dependent manner with higher doses leading to necrosis. Based on the lack of tumor inhibition in the monotherapy groups, it can be noted that tumors treated with PDT alone and Erbitux alone induced limited apoptosis in bladder carcinoma tumors. Therefore in this investigation, it was observed that the combination therapy significantly increased tumor cell apoptosis and inhibited tumor progression. Preclinically, many studies have shown that treatment with Erbitux in combination with radiotherapy or chemotherapy enhances apoptotic cell death than individual therapies. In a similar manner, PDT induced apoptosis, could have been enhanced by the combination of Erbitux to the treatment regime.

By using EGF phosphorylation antibody array membranes, we examined the relative level of phosphorylation of specific sites for human EGFR receptors. Interestingly, we noted the phosphorylation of Threonine 686 site of ErbB2 in all the groups. Studies have suggested that the dysregulation of cellular protein kinase C [46] and protein kinase A [47] activity could phosphorylate ErbB2 on Thr-686 for the activation and proliferation of tumor cells. However, our findings suggest that ErB2 on Thr-686 may not be essential for regulation of tumor proliferation, as tumor control was observed in the PDT + Erbitux treated group. Phosphorylation of EGFR tyrosine 845, only noticed in control tumors, is implicated in the stabilization of the activation loop, providing a binding surface for substrate proteins and is capable of regulating receptor function and tumor progression [48]. c-Src is known to be involved in the phosphorylation of EGFR at Tyr845 [49]. The major autophosphorylation sites of ErbB2 are Tyr1248 and Tyr1221/1222 that lead to Ras-Raf-MAP kinase signal transduction pathway [50]. In control tumors, ErbB2 was phosphorylated at tyrosine 1221/1222 and is associated with high tumor grade and with shorter disease-free survival and overall survival [51]. Similarly, ErbB4 is able to induce phosphorylation of phosphatidylinositol 3-kinase regulatory subunit which is a pro-survival protein that prevents apoptosis [52, 53]. Our data suggests that dephosphorylation of ErbB4 tyrosine 1284 is critical for tumor regression in the dual treatment group.

EGFR-mediated Ras-Raf-MEK-ERK and PI3K-PTEN-AKT pathways plays an important role in transmission of signals from membrane receptors to downstream targets that regulate apoptosis, cell growth and angiogenesis. Components of these pathways include genes such as Ras, B-Raf, PI3K, PTEN and Akt that can be mutated or aberrantly expressed in human cancer. Though we did not investigate these genes, it should be noted that they could cause resistance to anti-EGFR therapy. Numerous studies have reported Kras mutations as a predictor of resistance to Erbitux therapy and are associated with poor prognosis in colorectal cancer [54] and non-small cell lung carcinoma [55]. In a similar way, Braf mutation is also known to cause resistance to anti-EGFR therapy in colorectal cancers [56] and primary lung adenocarcinomas [57]. Mutation of PTEN tumor suppressor gene in human cancer cells leads to activated EGFR downstream signaling including PI3-kinase/AKT and have been linked to resistance to anti-EGFR targeted therapies [58]. However, in this study we investigated the role of EGFR target genes cyclin D1 and c-myc that are involved in cell proliferation. Our RT-PCR results showed downregulation of cyclin D1 and c-myc in the tumors treated with the combination therapy. Amplification of cyclin D1, a key cell cycle regulatory protein, appears to be an important event in bladder cancer and is often associated with cell proliferation and poor prognosis in human tumors [59]. In our study, downregulation of EGFR also resulted in reduction of cyclin D1. This observation could be due to the administration of Erbitux, that is known to cause cell cycle arrest in the G(1)/G(0)-phase, and also increases the expression of cyclin-dependent kinase inhibitors [60]. c-myc, another EGFR target gene that can obstruct the induction of apoptosis in tumor cells and lead to uncontrolled cell growth was reduced in the PDT plus Erbitux treated tumors. Over-expression and amplification of c-myc can play an important role in metastatic progression that indicates poor prognosis in different cancers [61]. These results suggest that EGFR target genes could play a role in tumor inhibition in bladder cancer by arresting cell cycle growth and inducing apoptosis.

In conclusion, combination treatment of PDT and Erbitux can improve the tumor response of bladder carcinoma xenografts. In this study, we observed that PDT induced tumor destruction can be maintained and significantly enhanced by the administration of Erbitux. As PDT treated tumors have been shown to adapt to inflammation and vascular shutdown, and PDT alone may not be sufficient for effective treatment, there is a need for combination of different modalities to obtain better tumor response. The challenge is to choose the appropriate anti-angiogenesis agent in combination with optimal PDT dosimetry for potential clinical application.