Involvement of the Integrin α1β1 in the Progression of Colorectal Cancer

Abstract

Integrins are a family of heterodimeric glycoproteins involved in bidirectional cell signaling that participate in the regulation of cell shape, adhesion, migration, survival and proliferation. The integrin α1β1 is known to be involved in RAS/ERK proliferative pathway activation and plays an important role in fibroblast proliferation. In the small intestine, the integrin α1 subunit is present in the crypt proliferative compartment and absent in the villus. We have recently shown that the integrin α1 protein and transcript (ITGA1) are present in a large proportion of colorectal cancers (CRC) and that their expression is controlled by the MYC oncogenic factor. Considering that α1 subunit/ITGA1 expression is correlated with MYC in more than 70% of colon adenocarcinomas, we postulated that the integrin α1β1 has a pro-tumoral contribution to CRC. In HT29, T84 and SW480 CRC cells, α1 subunit/ITGA1 knockdown resulted in a reduction of cell proliferation associated with an impaired resistance to anoikis and an altered cell migration in HT29 and T84 cells. Moreover, tumor development in xenografts was reduced in HT29 and T84 sh-ITGA1 cells, associated with extensive necrosis, a low mitotic index and a reduced number of blood vessels. Our results show that α1β1 is involved in tumor cell proliferation, survival and migration. This finding suggests that α1β1 contributes to CRC progression.

Keywords: ITGA1; cell migration; colorectal cancer; integrin α1; proliferation; survival.

Figure 1

Downregulation of the α1 integrin subunit in colorectal cancer cells. HT29, T84 and SW480 cells were infected with lentiviruses encoding a non-targeting short hairpin RNA (sh-ctl) or with shRNA targeting the ITGA1 mRNA (sh-ITGA1). Cells were selected with puromycin (10 μg/mL) 10 days before protein or RNA extraction. (A) Expression of the transcript of the ITGA1 gene was quantified by qPCR and normalized to the expression of the endogenous gene RPLP0. (B) Representative Western blot showing expression of the integrin α1 and α2 subunits in sh-ITGA1 cells compared to sh-ctl cells and densitometric analysis of the α1 subunit. Expression of ACTB was used as the protein loading control. Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Involvement of the integrin α1β1 in the proliferation of colorectal cancer cells. (A) Growth curves showing the cell counts up to 8 days after seeding of HT29, T84 and SW480. The curves show the number of live cells in the two groups; sh-ctl (black) and sh-ITGA1 (gray). Initially, 50000 cells were seeded into 6-well plates, and cells were counted every two days. (B) Histogram showing the results of 5-bromo-2′-deoxyuridine (BrdU) incorporation into the cells, performed 4 days after seeding of the same three cell lines. In each field, labeled nuclei were counted and compared to the total number of nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI). The experiments were performed in triplicate and were repeated three times. Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Involvement of integrin α1β1 in anoikis resistance in colorectal cancer. Histograms showing the results of the measurement of caspase-3 activity in the three cell lines HT29, T84 and SW480. The cells were kept in suspension without serum for 24 h in plates coated with poly 2-hydroxyethyl methacrylate. Cells were lysed using buffer without p-nitrophenyl phosphate. N = 3. Student’s t test. * p < 0.05.

Figure 4

Involvement of the integrin α1β1 in cell migration. (A) Schematic representation of the scratch assay procedure. Cells were plated at high density and treated with 2 mM hydroxyurea for 24 h before and throughout the assay. Two to three wounds were made in each Petri dish (red lines). A line (in black) was drawn under the Petri dish to serve as a landmark to track cell migration. (B) Representative micrographs of the scratch assay for HT29 and T84 cell lines. The evolution of the closure of the wounded area was compared between sh-ITGA1 and sh-ctl groups from 0 to 48 h. (C) Histogram showing the result of the initial area created by the injury (0 h) with respect to the final surface (48 h). The experiments were performed in triplicate and were repeated three times. Student’s t test. * p < 0.05. Scale bars = 100 μm.

Figure 5

Integrin α1 subunit/ITGA1 knockdown reduced the development of colorectal tumors in xenografts. (A) Tumor growth assessment after injection of 2 × 10⁶ HT29 or T84 cells into the subcutaneous tissue of nu/nu mice. For both cell lines, the sh-ctl and sh-ITGA1 cells were injected after 10 days of selection with puromycin (10 µg/mL). Tumors were measured in two axes and the volume was determined using the formula V = (D × d2)/2. (B) Representative micrographs of the histological architecture of the tumors derived from sh-ITGA1 and sh-ctl cells for HT29 and T84 cell lines. Hematoxylin and eosin staining (H&E). (C,D) Representative Western blot and immunohistochemical micrographs showing the validation of the repression of expression of the α1 subunit, after resection of tumors derived from sh-ctl and sh-ITGA1 cells, for HT29 and T84 cell lines. N = 3. Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar = 100 µm.

Figure 6

Suppression of the expression of the integrin α1 subunit/ITGA1 results in fewer mitotic cells, fewer blood vessels and increased necrosis in xenografts. (A) Representative micrograph of tumor sections from HT29 sh-ctl and sh-ITGA1 cells stained with H&E. Mitotic cells are surrounded by a red circle. (B) Histogram showing the number of mitotic cells per mm² in both sh-ctl and sh-ITGA1 tumors. For each tumor, four areas were randomly selected and analyzed for mitotic count. (C) Representative immunohistochemical staining showing intratumoral capillaries surrounded by a red circle. Endothelial cells were identified using anti-CD31 antibodies as shown in the red box. (D) Results of the count of the number of capillaries in the sh-ctl and sh-ITGA1 tumors. (E) Representative micrograph of sh-ctl and sh-ITGA1 HT29-derived tumors. Tumors were cut along the longest axis and then sections were stained with H&E. For each tumor, the necrotic eosinophilic areas are outlined in red. (F) Histogram showing the result of the percentage of necrotic areas compared to the total area of each tumor. The results represent the average analysis of three samples. Student’s t test. * p < 0.05, *** p < 0.001. Scale bar = 100 µm.