The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma

<div><p>Cancer stem cells (CSCs) play an important role in the clonogenic growth and metastasis of pancreatic ductal adenocarcinoma (PDAC). A hallmark of PDAC is the desmoplastic reaction, but the impact of the tumor microenvironment (TME) on CSCs is unknown. In order to better understand the mechanisms, we examined the impact of extracellular matrix (ECM) proteins on PDAC CSCs. We quantified the effect of ECM proteins, β1-integrin, and focal adhesion kinase (FAK) on clonogenic PDAC growth and migration <i>in vitro</i> and tumor initiation, growth, and metastasis <i>in vivo</i> in nude mice using shRNA and overexpression constructs as well as small molecule FAK inhibitors. Type I collagen increased PDAC tumor initiating potential, self-renewal, and the frequency of CSCs through the activation of FAK. FAK overexpression increased tumor initiation, whereas a dominant negative FAK mutant or FAK kinase inhibitors reduced clonogenic PDAC growth <i>in vitro</i> and <i>in vivo</i>. Moreover, the FAK inhibitor VS-4718 extended the anti-tumor response to gemcitabine and nab-paclitaxel in patient-derived PDAC xenografts, and the loss of FAK expression limited metastatic dissemination of orthotopic xenografts. Type I collagen enhances PDAC CSCs, and both kinase-dependent and independent activities of FAK impact PDAC tumor initiation, self-renewal, and metastasis. The anti-tumor impact of FAK inhibitors in combination with standard chemotherapy support the clinical testing of this combination.</p></div

FAK overexpression increases PDAC clonogenic growth and tumor initiating capacity.

<p>(<b>a</b>) Ratio of phospho-FAK to total FAK expression by MIA PaCa2 cells expressing a scrambled control (shCtrl) or β1 integrin (shBeta1) shRNA and cultured on plastic or type I collagen for 96 hours. (<b>b</b>) Colony formation by MIA PaCa-2 and Capan-1 cells overexpressing FAK-FL following growth on plastic or type I collagen for 96 hours. Data represent mean ± SD (<i>n</i> = 4) of control versus FAK-FL; *<i>P</i> < 0.05; **<i>P</i> < 0.001; ***<i>P</i> < 0.0001. (<b>c</b>) Tumor growth of MIA PaCa2 cells overexpressing FAK-FL following subcutaneously injection in to NSG mice. *<i>P</i> = 0.04 compared to control (vector).</p

The loss of FAK inhibits PDAC cell migration <i>in vitro</i> and metastasis <i>in vivo</i>.

<p>(<b>a</b>) <i>In vitro</i> migration by MIA PaCa-2 and Capan-1 cells expressing scrambled control (shCtrl) or FAK shRNA following treatment with doxycycline on type I collagen for 96 hours. Data represent mean ± SD (<i>n</i> = 4) of control versus hairpin; **<i>P</i> < 0.001. (<b>b</b>) <i>In vivo</i> metastases by orthotopic MIAPaCa-2 tumors expressing scrambled control (shCtrl) or FAK shRNA (shFAK) following treatment with doxycycline. Seven mice were used in each group. Control versus hairpin; *<i>P</i> = 0.008.</p

FAK kinase-inhibition decrease clonogenic PDAC growth <i>in vitro</i> and inhibits tumor growth <i>in vivo</i>.

<p>(<b>a</b>) <i>In vitro</i> colony formation by MIA PaCa-2 and Capan-1 cells following treatment with vehicle control (DMSO) or VS-4718 on type I collagen for 96 hours. Data represent mean ± SD (<i>n</i> = 3) of DMSO versus VS-4718; *<i>P</i> < 0.05. (<b>b</b>) Colony formation by 2 distinct patient derived xenograft cells following treatment with vehicle control (DMSO) or VS-4718 on type I collagen for 5 days. (<b>c</b>) Subcutaneous tumor growth of a patient derived xenograft (JH102) following treatment with vehicle control, VS-4718, gemcitabine plus nab-paclitaxel (Gem-Pac), or all three drugs together (Gem-Pac-VS). Five mice were included in each group. **<i>P</i> = 0.0076, *<i>P</i> = 0.03 by ANOVA. (<b>d</b>) Colony formation by MIA PaCa-2 and Capan-1 cells overexpressing FAK-Y397F following growth on type I collagen for 96 hours. Data represent mean ± SD (<i>n</i> = 4) of control versus FAK-Y397F; *<i>P</i> < 0.05, **<i>P</i> < 0.001.</p

FAK regulates tumor initiating cell (TIC) frequency in PDAC.

<p>FAK regulates tumor initiating cell (TIC) frequency in PDAC.</p

The loss of FAK inhibits self-renewal.

<p>(<b>a</b>) Frequency of ALDH⁺ MIA PaCa-2 and Capan-1 cells expressing scrambled control (shCtrl) or FAK shRNA (shFAK) following treatment with doxycycline for 96 hours. Data represent mean ± SD (<i>n</i> = 3) of shCtrl versus shFAK; *<i>P</i> < 0.05. (<b>b</b>) <i>In vitro</i> colony formation by MIA PaCa-2 and Capan-1 cells expressing scrambled control (shCtrl) or FAK shRNA (shFAK) following treatment with doxycycline for 96 hours on type I collagen. Data represents ± SD (<i>n</i> = 4) of control versus hairpin; **<i>P</i> < 0.001. (<b>c</b>) <i>In vivo</i> subcutaneous tumor growth by MIA PaCa-2 cells expressing scrambled control (shCtrl, n = 4) or FAK shRNA (shFAK, n = 7) following treatment with doxycycline. Error bar represents SD; *<i>P</i> = 0.02.</p