This is the first study of PINCH expression in rectal cancer patients who participated in a Swedish clinical trial of preoperative RT. In patients without RT, strong PINCH expression at the invasive margin of primary tumours was related to worse survival compared to patients with weak PINCH expression. The prognostic significance still remained even after adjustment for both TNM stage and differentiation. This result was in line with others who studied PINCH expression in 174 colorectal cancer patients [7
]. After RT, there was no relationship between PINCH and survival. A further interaction analysis showed no statistically significant result, which might indicate that the number of deaths in the RT group was low. As far as we know, this is the first study of PINCH in relation to RT in patients. A previous cell line study of mouse embryonic fibroblasts and human colon, lung, cervix, skin and pancreas tumours showed that PINCH was radio-resistant by activating Akt1 [10
]. Others showed that the radio sensitivity in PINCH depleted normal and malignant cells was similar under adherent and suspension conditions [11
]. In this study, we did not found any significant difference in PINCH expression between the subgroups of non-RT and RT, which might be explained by a too short interval between RT and surgery. The best clinical effects of RT on tumour tissue are known to be received around 5 weeks after RT. In our study, the patients received preoperative RT and went through surgery within 1-13 days after RT, which might be a too short time to receive the optimal clinical effect by RT. Even though there was no significant difference in PINCH expression between the non-RT and RT subgroups, the survival for patients with weak and strong PINCH expression seemed to change with RT, which makes us suggest that PINCH might not be directly increased by RT, but maybe activated by RT via other biological pathways.
PINCH together with its binding partners are known to regulate cell survival and apoptosis [4
]. Previously, it was shown that PINCH induced radio-resistance by activating Akt1 via PP1α [10
]. Others showed that an inhibition of the PINCH-ILK complex increased apoptosis by reducing the activity of protein kinase B (PKB)/Akt in immortalised HeLa cells [4
The epidermal growth factor (EGF) is a well-known growth factor, which is mitogenic and stimulates cell division by binding to a tyrosine kinase receptor on the cell membrane. RT is known to up-regulate the EGF receptor [18
]. Recently, it was shown that EGF together with its receptor was associated with PINCH via the adaptor protein Nck-2 [4
]. RT induced cell damage might increase the production of EGF witch further activates PINCH via Nck-2. We suggest that the cell damage that RT causes could be the initiating mechanism, not for an up-regulation of PINCH, but for an activation of PINCH, via the PKB/Akt pathway or EGF and Nck-2 pathway.
The lymphatic vasculature drains interstitial fluid from tissue and is one of the most common ways for tumour cells to metastasis and spread. Recently, it was shown that LVD was increased by RT [20
]. In the present study of the patients with RT, a positive relationship was found between PINCH at the inner tumour area and LVD. We suggest that PINCH might stimulate the production of new lymph vessels as a reaction to RT induced cell damage. The positive relationship between PINCH and LVD after RT, might increase the area for potential escape of tumour cells into the lymphatic circulation.
In line with our previous findings, our present study of the fibroblast cell line showed no changes in PINCH expression after RT. PINCH are known to be widely expressed in fibroblasts and increases from normal mucosa to tumour [6
]. The cells used in our study are supposed to be normal fibroblasts with less expression of PINCH than the fibroblasts in the tumour tissue. Since we did not find any differences in PINCH expression in tumours after RT, the probability to find differences in PINCH expression in the fibroblast cells after RT might be low.
The infiltration of inflammatory cells in tumour tissue is considered as an important factor of the host response, and is related to improved survival in colorectal cancer [14
]. A recent study on colorectal cancer patients showed that a high PINCH expression was related to weak inflammatory infiltration [7
]. In line with this study, at the inner tumor area and invasive margin, in all patients and in the non-RT subgroup, we found a relationship between strong PINCH expression and weak inflammatory infiltration. Gao et al. (2004) showed an increased amount of PINCH in myofibroblasts suggesting that these cells induce the tumour reaction against inflammatory cell infiltration.
Apoptosis is programmed cell death and decreased apoptosis are related to worse survival in tumours. Previously, it was shown that PINCH inactivated the intrinsic apoptotic pathway [12
]. Ours found the same result in non-irradiated patients, where a strong PINCH expression at the invasive margin was related to less apoptosis. These findings strengthen our previous relationships between strong PINCH expression and less inflammatory infiltration and worse survival. We suggest that PINCH at the invasive margin might facilitate tumour progression and survival by inhibiting inflammatory infiltration and reduce apoptosis.
The relationship between BVD and survival has been studied extensively. In a previous study by ours on the same series of the cases used in the present study, patients in the non-RT subgroup with BVD at the periphery tended to have a worse outcome than the patients with BVD at the inner tumour area/invasive margin [14
]. In the present study of all patients and in patients without RT, PINCH expression at the inner tumour area was related to a higher BVD at the periphery of tumours. At the periphery, PINCH might create an environment that makes it easier for tumour cells to transit into the blood system.
Tumour necrosis is caused by a rapid tumor growth without sufficient blood supply, which leads to ischemia and necrosis of the tumour cells. Previous studies by others did demonstrate that necrosis was associated with a poor clinical outcome [21
]. In the present study of all patients, we found a positive relationship between PINCH at the invasive margin and necrosis. Since PINCH is involved in cell regeneration we suggest that PINCH might induce cell proliferation which further leads to un-sufficient blood supply, ischemia and necrosis of the tumour tissue.
In a previous study by ours on the same series of the cases used in the present study, necrosis was increased by RT [15
]. In the present study, in patients with RT, a strong PINCH expression was positively related to more necrosis. PINCH seems to be involved in the initiation of necrosis induced either by rapid tumour growth or by RT.
In this material the surgery was performed either by anterior resection or rectum amputation. In the 1980th a new surgical technique called total mesorectal excision (TME) was introduced, which was shown to reduce the risk of local recurrence up to 11% and increased the overall survival up to 40%. The combination of preoperative RT with TME further reduced the local recurrence rate up to around 5% [22
]. Even though the local recurrence rate has been reduced the mortality rate is still high (40-50%) and there are still huge variations in response to preoperative RT in patients with the same tumour stage. Therefore it is of great importance to identify good predictive and prognostic factors such as PINCH in order to select the best suited patients for preoperative RT in the future.