Immunohistochemical expression of mitochondrial membrane complexes (MMCs) I, III, IV and V in malignant and benign periampullary epithelium: a potential target for drug therapy of periampullary cancer?

The mitochondrial respiratory chain complexes or mitochondrial membrane complexes (MMCs) I, II, III and IV are proteins responsible for electron transport and the associated proton pumping which generates a proton gradient and mitochondrial membrane potential which is then used (via ATP synthase, complex V) to generate adenosine-5'-triphosphate (ATP), the central energy currency of the cell. Mitochondria also play a key role in apoptosis, through the modulation of membrane potential and the co-ordinated release of mitochondrial proteins such as cytochrome c [1]. Mitochondrial complexes have also been shown to be specific targets for drug therapy in cancer and drugs called "mitocans" [2–5] can disrupt the integrity of mitochondria causing cytosolic release of modulators of apoptosis and activation of mitochondria-dependent cell death signalling pathways inside cancer cells [6–8].

Although the concept of mitocans as novel anticancer agents is interesting, their safety and efficacy are dependent on their selectivity in targeting malignant tissue over normal tissue in inducing apoptosis [7, 9–11]. A key approach to targeting mitochondrial components involves blocking the electron transport chain by inhibiting MMCs I, II, III, IV and V [11, 12]. What is key to effecting some paradigms of mitochondrially-mediated apoptosis is the ability to induce a sufficiently large decrease in mitochondrial membrane potential, which may be made possible by inhibiting any one specific mitochondrial membrane.

Several classes of drugs are capable of achieving this in vitro [12–15], but before these agents are investigated clinically in specific cancers it is critical to ascertain the relative expression of these target membrane complexes in malignant tissue compared to benign tissue. Such a mechanistic approach would enable selection of mitocans that are target specific with least toxicity and optimal efficacy.

Periampullary cancer is a collective term used for cancers arising from or near the ampulla of Vater and include ampullary cancers, duodenal cancers, pancreatic head cancer and distal cholangiocarcinoma. As the relative expression of MMCs in malignant and benign pancreatic epithelium has not yet been characterized, we performed this study to determine the immunohistochemical expression of MMCs in tissue microarrays (TMAs) of formalin-fixed archived specimens of periampullary cancers (pancreatic ductal adenocarcinoma, ampullary adenocarcinoma, cholangiocarcinoma and duodenal adenocarcinoma), chronic pancreatitis, and normal pancreatic tissue, and to determine any association of MMC expression in cancer with survival.

Of the 126 consecutive patients who underwent pancreatic resections 104 (62 male) with a median (IQR) age of 64 (59-72) years had cancer and 22 (15 male) with a median (IQR) age of 55 (41-61) years had chronic pancreatitis. Specimens of chronic pancreatitis tissue were obtained from areas adjacent to cancer in 78 patients. Normal pancreatic tissue was obtained from the resection specimens, adjacent to areas of cancer or chronic pancreatitis in a total of 30 patients. Sixty-one metastatic lymph nodes from 61 patients were also studied [16]. Of the 104 patients with cancer, 31 (15 male) had ampullary adenocarcinoma, 24 (15 male) had cholangiocarcinoma, 44 (29 male) had pancreatic ductal adenocarcinoma and 5 (3 male) had duodenal adenocarcinoma. The ratios of well:moderately:poorly differentiated tumours were 7:16:8, 1:18:5, 5:28:11 and 0:5:0 for ampullary adenocarcinoma, cholangiocarcinoma, pancreatic ductal adenocarcinoma and duodenal adenocarcinoma respectively. Vascular invasion was present in 13 (42%), 8 (33%), 22 (50%) and 1 (20%) patients with ampullary adenocarcinoma, cholangiocarcinoma, pancreatic ductal adenocarcinoma and duodenal adenocarcinoma respectively. Corresponding figures for perineural invasion were 12 (39%), 14 (58%), 30 (68%) and 2 (40%) and those for lymphatic invasion were 12 (39%), 14 (58%), 26 (59%) and 4 (80%). Tumours were ≥ 2 cm in diameter in 12 (39%), 7 (29%), 15 (34%) and 2 (40%) patients with ampullary adenocarcinoma, cholangiocarcinoma, pancreatic ductal adenocarcinoma and duodenal adenocarcinoma respectively. AICC disease stage distribution (Ia:Ib:II) was 12 (39%):11 (35%):8 (26%) for those with ampullary adenocarcinoma, 7 (29%):7 (29%):10 (42%) for cholangiocarcinoma, 15 (34%):12 (27%):17 (39%) for pancreatic ductal adenocarcinoma and 2 (40%):2 (40%):1 (20%) for duodenal adenocarcinoma.

MMC expression by tissue type in the same tumour and by diagnosis

MMC I expression in cancer was not significantly higher than in benign tissue. A significantly greater expression of MMCs III, IV and V was noted in cancer cells compared with surrounding benign tissue (reactive pancreatitis or normal pancreas) (Figures 1, 2, 3 and 4). The pattern of expression is summarized in Figure 5. In contrast to differential expression within tissues of the same tumour, MMC expression across a range of periampullary cancer types showed an increased expression in all of them compared with chronic pancreatitis MMCs III, IV and V (Figure 6).

There was a tendency to better survival in patients who showed high expression of MMC I in their cancer compared with those who had no or low expression of MMC I, although this did not reach statistical significance (Kaplan Meier, Mantel Cox p = 0.08). However, higher expression of MMC V in the cancer, was associated with better survival (Kaplan Meier, Mantel Cox p = 0.005). This may, in part, relate to lower expression of these MMCs in poorly differentiated tumours compared to well and moderately differentiated tumours (Table 1, Figure 7). In addition, high expression of MMC V showed inverse correlation with lymph node metastases (r = -0.314, p < 0.001) and high VCMI (r = -0.231, p = 0.018).

This study has shown that MMCs I, III, IV and V are highly expressed in periampullary cancers compared with metastatic cancer in lymph nodes or benign periampullary tissue for MMCs III, IV and V. MMC II expression was not studied because of non-availability of a suitable monoclonal antibody for immunostaining of formalin-fixed tissues. Normal ductal cells exhibit very low levels of staining, which may be to due to low levels of mitochondrial expression in these cells, and/or the limited sensitivity of the methods/antibodies used. This, to the best of our knowledge, is the first study demonstrating a differential expression of MMCs in malignant compared with benign periampullary tissue. In future studies, we will also correlate our data with the expression of the tricarboxylic acid (TCA) cycle (Krebs' cycle) enzyme citrate synthase which has been used as a marker of mitochondrial mass and may allow detection of mitochondria in ductal cells due to its high level of expression in normal cells. Cancer cells are capable of withstanding an adverse microenvironment (hypoxia, acidosis, hypoglycaemia, and shortage of growth factors) by virtue of metabolic adaptation [19]. MMCs I and III, in addition to their role as proton carriers in the electron transport chain, are known to be crucial mediators for hypoxia induced reactive oxygen production [20–22]. Such a mechanism is crucial for cellular survival in adverse hypoxic conditions. Therefore, a differentially higher expression of MMCs in primary periampullary cancer, compared with metastatic lymph nodes or benign periampullary tissue may represent an adaptive mechanism to chronic cellular hypoxia.

Periampullary cancers carry a poor prognosis due to their insensitivity to most conventional therapies [23, 24]. Currently, surgical resection offers the only potential chance for a cure in these patients. The 5-year survival rate of all patients with pancreatic cancer is below 5%, and the median survival time after diagnosis is 6 months and only about 20% of patients who undergo curative resection survive longer than 5 years [25].

One mechanism of action of mitochondrial targeted anti-cancer drugs relies on their ability to disrupt the energy producing systems of cancer cell mitochondria, leading to increased reactive oxygen species and activation of the mitochondrial dependent cell death signaling pathways inside cancer cells. This emerging class of drugs called "mitocans" should act by altering properties of the mitochondria inside cancerous but not normal cells for instance by reducing the resting membrane potential by blocking the electron transport chain. It is clear from the present studies that mitocans offer great potential as effective and exciting new developments in cancer therapy, providing direct activation of cancer cell death by mitochondrial mediated apoptosis and that this complements the other pathways by which existing treatments kill cancer cells [2–5]. A proof of concept for MMC targeted therapy is the ability of clomipramine induced inhibition of MMC-III in cancer cells, as previous shown by our group [12]. Vitamin E analogues [4] have been shown to be an inhibitor of MMC II and tamoxifen, an inhibitor of MMC I [26].

Mitochondria-mediated anti-cancer therapy may also rely on manipulation of tumour suppressor proteins that are encoded by mitochondrial membrane complex genes. These genes include the succinate dehydrogenase (SDH) encoder. SDH is part of the TCA cycle that connects glucose metabolism in the cytosol to oxidative phosphorylation in the mitochondria and is the only membrane-bound enzyme of the TCA cycle and is also a functional member (complex II) of the electron transport chain. Inherited or somatic mutations in SDH have been shown to lead to the development of phaeochromocytomas and paraganglionomas [27].

However, the clinical safety and efficacy of potential anti-mitochondrial therapies in periampullary tumours lies in the selective targeting of malignant epithelium with relative sparing of benign tissue. The prudent choice of a MMC targeting agent would depend on its differential expression between malignant and benign tissue.

Enhanced expression of MMCs III and V in tumours compared with chronic pancreatitis supports their mechanistic safety when administered to diagnostically uncertain pancreatic cancers, which may turn out to be chronic pancreatitis. Moreover, high expression of some of these MMCs (I and V) may be associated with better survival, by suppressing tumour biological factors like proliferative capacity and lymph node metastasis.

MMC III, IV and V targeting in periampullary cancers is mechanistically viable based on their characteristic expression profiles, indicating that such targeting strategies can be selective and therefore likely to be effective with minimal collateral damage to benign pancreatic tissue.