AbstractLaminin binding integrins, A3B1 and A6B1, drive in part cancer cell migration and metastasis. Cell migration requires dynamic internalization and recycling of surface integrins. However, whether laminin binding integrin internalization is coordinately or independently controlled is not known. In this study, we quantitated the internalization of integrin A3 and A6 and whether crosstalk influences their respective internalization and subsequent cancer cell migration.We quantitated the internalization of A6 and A3 integrins, using fluorophore conjugated antibody based internalization assays and flow cytometry. In the prostate cancer cell line DU145, we observed constitutive internalization of the integrin A6 with an actual rate constant (kactual) of 3.25min-1 as compared to 1.00min-1 for integrin A3, 2.20min-1 for unrelated integrin Av or 15.08min-1 for the unrelated transferrin receptor. Silencing A3 integrin expression resulted in an 11 percent increase in the total amount of internalized A6 integrin, with a 1.5 fold increase in the actual rate constant. The internalized A6 integrin showed intracellular punctate staining colocalized with the early endosome marker EEA1 and Rab4 containing vesicles. Silencing A3 integrin led to 1.8 fold increase in migration which was dependent on A6B1 integrin. Silencing integrin A6 expression, however, did not affect the internalization of A3 integrin indicating a unidirectional regulation of integrin internalization. The changes in the integrin internalization could be clearly ascribed to alpha subunits, as the obtained internalization kinetics of A6 and A3 integrins were independent of the antibody used or integrin engagement by ligand mimetic peptides. Taken together, these data indicate that A6B1 and A3B1 integrins have different internalization kinetics and the presence of integrin A3 decreased integrin A6 internalization and ensuing cell migration. These data are consistent with previous observations in normal systems suggesting that A3B1 provides a provisional matrix during early stages of wound healing, requiring subsequent stable adhesion complexes and integrin A6 function. In cancer, the loss of A3 integrin and the increased internalization of A6 integrin may account for the "wounds that won't heal" phenotype and provide a strategy for biological intervention to block tumor progression.(Supported in part by NIH Grants CA23074 and CA159406)

BackgroundLaminin binding integrins A6 and A3 are persistently expressed in prostate cancer and bone metastases. Loss of A3 expression is observed in a subpopulation of high Gleason grade carcinoma (1).

• A6 integrin expression is associated with poor patient prognosis, reduced survival and increased metastasis in a variety of tumors.

• A6 integrin is cleaved by urokinase plasminogen activator to promote invasion and metastasis of cancer (2).

• A6 integrin internalization and trafficking by small RabGTPases drives migration of highly motile neural cells (3) and hypoxia induced cancer (4).

Lipsa Das1, Todd A. Anderson1, Jaime M.C. Gard1, Isis C. Sroka Ph.D.1, Stephanie R. Strautman1, Raymond B. Nagle M.D, Ph.D.1, Beatrice S. Knudsen M.D., Ph.D.2, Anne E. Cress Ph.D.1

1The University of Arizona Cancer Center, College of Medicine, University of Arizona, Tucson, AZ, 2Cedars Sinai Medical Center, Los Angeles, CA.

Results

Figure 2: Internalization rates of integrins A3 and A6 are distinct from B1 and other alpha integrin subunit. (A) Schematic representation of the quantitative internalization assay. (B) Flow cytometry histogram of internalized label against A6, A3, Av, B1 integrins or transferrin receptor. (C) Internalization kinetics curve plotted using the percent of label internalized (calculated using the mean peak fluorescence values at each timepoint divided by the full label value and multiplied by 100). First order kinetic curve fitting was used to calculate the observed and actual rate constant of internalization (kobs, kactual)and maximum surface receptor availability for internalization (amplitude, b).

N PNICA

*

V

Figure 1: A6 integrin is constitutively internalized in invasive prostate cancer and bone metastases. Human prostate cancer (A) and its bone metastases (B) was reacted with antibody specific for A6 integrin (AA6NT) as previously described (2). Both membrane and intracellular localization of A6 integrin can be seen in cancer (CA), invasive budding structures (*) and perineural invasion (PNI). Nerve (N) and blood vessels (V) are positive for A6 as expected. (C) Constitutive internalization of integrin A6 and its cleaved variant A6p in DU145 prostate cancer cell line. Cell surface A6 integrin was labelled with biotin and allowed to internalize for different time intervals. Membrane bound biotin was removed, cell lysate was immunoprecipitated with J1B5 and analyzed by western blot.

Prostate Cancer

A

Bone Metastases

A6

A6p

CB

225

150

102

76

A B C

Figure 5: Silencing A3 redistributes A6 by early endosome and Rab4 mediated intracellular trafficking. DU145 cells were surface labelled with anti-A6 antibody, J1B5 (red) and allowed to endocytose A6 for 45 mins. Cells were fixed, permeabilized and immunostained for early endosome marker EEA1 (green) and recycling vesicles Rab4 (blue). Increased co-localization of the integrin A6 with EEA1 and Rab4 was observed in ITGA3 silenced cells (bottom panel) as compared to untreated (top) and siControl treated (middle) cells. Images acquired by confocal microscopy. Bars, 20um.

Figure 3: Laminin receptors crosstalk regulates A6 internalization. DU145 prostate cancer cells were depleted of the A3 or A6 integrin using siRNA (siA3 and siA6, respectively) for 72 hrs and compared to wild type control (WT) and non-targeting control (siCON). Flow cytometer profile of internalization (A) and internalization kinetic curve analysis of A6 on silencing A3 (B). Flow cytometer profile of internalization (C) and internalization kinetic curve analysis of A3 on silencing A6 (D). Western blot analyses showing successful silencing of respective alpha subunits (E).

Figure 4: Internalization kinetics of A3 and A6 subunits are unaffected by B1 integrin antibody or ligand mimetic. (A) DU145 cells treated with the B1 integrin specific antibody A2BII. (B) DU145 cells treated with A6 integrin receptor ligand mimetic HYD1 and HYDS, a scrambled peptide control.

A B

E

Figure 6: Silencing A3 promotes prostate cancer cell migration dependent on A6 integrin and its cleavage. (A) Silencing of A3 significantly increased DU145 cell migration which was inhibited by B1 blocking antibody (A2BII). (B) Silencing of A6 and functional blocking of A6 cleavage by anti-A6 antibody (J8H) decreased DU145 cancer cell migration (white bars). A3 silencing induced increase in cell migration was blocked by dual depletion of A6 and functional blocking of A6 cleavage J8H antibody (black bars).

A B

References1.Schmelz M, Cress AE, Scott KM, Bürgery F, Cui H, Sallam K, McDaniel KM, Dalkin BL, Nagle

RB. Different Phenotypes in Human Prostate Cancer: α6 or α3 Integrin in Cell-extracellular Adhesion Sites. Neoplasia. May 2002; 4(3): 243–254.

2.Ports MO, Nagle RB, Pond GD, Cress AE. Extracellular Engagement of α6 Integrin Inhibited uPA Mediated Cleavage and Delayed Human Prostate Bone Metastasis. Cancer Res. 2009; 69(12): 5007–5014.

3. Stachan LR and Condic ML. Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility. J Cell Biol. 2004; 167(3): 545-54.

4.Yoon SO, Shin S, Mercurio AM. Hypoxia stimulates carcinoma invasion by stabilizing microtubules and promoting the Rab11 trafficking of the a6b4 integrin. Cancer Res. 2005; 65(7):2761-9.

5. Sroka IC, Anderson TA, McDaniel KM, Nagle RB, Gretzer MB, Cress AE. The laminin binding integrin alpha6beta1 in prostate cancer perineural invasion. J Cell Physiol.2010; 224(2):283-8.

6.Zhou B, Gibson-Corley KN, Herndon ME, Sun Y, Gustafson-Wagner E, Teoh-Fitzgerald M, Domann FE, Henry MD, Stipp CS. Integrin α3β1 can function to promote spontaneous metastasis and lung colonization of invasive breast carcinoma. Mol Cancer Res. 2014 Jan;12(1):143-54

Conclusions• A6 presents both cell membrane and intracellular expression in

invasive prostate cancer and bone metastasis (Fig.1).• Integrins are constitutively internalized in cancer cells where A6 has a

3 fold higher rate constant of internalization than A3 integrin (Fig.2).• A6 integrin internalization is independent of partner B1 subunit and

ligand binding (Fig.4).• Loss of A3 in cancer

• increases internalization of A6 (Fig. 3)• promotes accumulation of A6 in early endosomes and recycling

by Rab4 vesicles to the cell-cell lateral membrane (Fig. 5)• increases tumor cell migration by 1.8 fold which is dependent on

A6 integrin function and cleavage (Fig. 6).

A6B1 Integrin Internalization is Increased by Loss Of A3B1 Expression and Promotes Migration of Human Prostate Cancer Cells.