Loss of RhoA Exacerbates, Rather Than Dampens, Oncogenic K-Ras Induced Lung Adenoma Formation in Mice

<div><p>Numerous cellular studies have indicated that RhoA signaling is required for oncogenic Ras-induced transformation, suggesting that RhoA is a useful target in Ras induced neoplasia. However, to date very limited data exist to genetically attribute RhoA function to Ras-mediated tumorigenesis in mammalian models. In order to assess whether RhoA is required for K-Ras-induced lung cancer initiation, we utilized the K-Ras^G12D Lox-Stop-Lox murine lung cancer model in combination with a conditional RhoA^flox/flox and RhoC^-/- knockout mouse models. Deletion of the floxed <i>Rhoa</i> gene and expression of K-Ras^G12D was achieved by either CCSP-Cre or adenoviral Cre, resulting in simultaneous expression of K-Ras^G12D and deletion of RhoA from the murine lung. We found that deletion of RhoA, RhoC or both did not adversely affect normal lung development. Moreover, we found that deletion of either RhoA or RhoC alone did not suppress K-Ras^G12D induced lung adenoma initiation. Rather, deletion of RhoA alone exacerbated lung adenoma formation, whereas dual deletion of RhoA and RhoC together significantly reduced K-Ras^G12D induced adenoma formation. Deletion of RhoA appears to induce a compensatory mechanism that exacerbates adenoma formation. The compensatory mechanism is at least partly mediated by RhoC. This study suggests that targeting of RhoA alone may allow for compensation and a paradoxical exacerbation of neoplasia, while simultaneous targeting of both RhoA and RhoC is likely to lead to more favorable outcomes.</p></div

Breeding Schematic for Transgenic Mice.

<p>Breeding Schematic for Transgenic Mice.</p

RhoA is not essential for CCSP-promoter driven, K-Ras^G12D-induced, lung adenoma formation in mice.

<p>Rho^WT and RhoA^flox/flox mice were mated with CCSP-Cre mice on a K-Ras^G12D background. <b>(A & B)</b> K-Ras^G12D lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively). <b>(C)</b> Immunohistochemistry for pERK^{Thr202/Tyr204} (brown) with hematoxylin counterstain (bars represent 100μm). <b>(D)</b> Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent 100μm). <b>(E)</b> Quantification of the RhoA-status of adenomas as assessed by immunohistochemistry. Greater than 30 tumors were counted from four mice per group.</p

Neither RhoA nor RhoC is required for K-Ras^G12D-induced adenoma formation in a CCSP-Cre model.

<p>Rho^WT, RhoC^-/- and DKO mice were mated with CCSP-Cre mice on a K-Ras^G12D background. <b>(A & B)</b> K-Ras^G12D lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively). <b>(C)</b> Immunohistochemistry for pERK^{Thr202/Tyr204} (brown) with hematoxylin counterstain (bars represent 100μm). <b>(D)</b> Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent 100μm). <b>(E)</b> Quantification of the RhoA-status of adenomas as assessed by immunohistochemistry. Greater than 30 tumors were counted from four mice per group.</p

RhoA and RhoC are important in combination, but not individually, for K-Ras^G12D induced sporadic lung adenoma formation.

<p>Mice from different Rho backgrounds were administered Adeno-Cre virus endotracheally. Lungs were harvested after 12 weeks. <b>(A)</b> H&E of adenomas (bars represent 100μm). <b>(B)</b> Immunohistochemistry for pERK^{Thr202/Tyr204} (brown) with hematoxylin counterstain (bars represent 100μm). <b>(C)</b> Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent 100μm). <b>(D)</b> Western blots of microdissected tumors (N = normal lung, T = tumor). <b>(E)</b> Quantification of tumor burden. Means represent the quantification of four separate, evenly spaced and similarly sized lung sections, from four mice for each group (n = 4, * = p ≤ 0.001). Data representative of two separate experiments. <b>(F)</b> Quantification of tumor sizes. Data points represent the area of individual tumors expressed in μm². Red bars represent the mean tumor area. <b>(G)</b> Proportion of RhoA-positive and RhoA-null tumors. Displayed as RhoA staining by the level of staining intensity (levels are as follows: no staining; normal = same intensity staining as adjacent normal alveoli; high = greater than adjacent normal alveoli; very high = much greater intensity staining than adjacent normal alveoli).</p

CCSP-promoter driven deletion of RhoA does not affect normal lung development.

<p>Rho^WT and RhoA^flox/flox mice were mated with CCSP-Cre mice on a K-Ras^WT background. <b>(A & B)</b> Lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively). <b>(C)</b> Mice were crossed to a tdTomato to eGFP reporter line to assess for difference is Cre-expression pattern (bars represent 100μm). Green represents eGFP and Cre activity. Red presents tdTomato and the absence of Cre activity. Blue represents DAPI staining. <b>(D)</b> Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent 25μm). <b>(E)</b> Immunohistochemistry for CCSP (black), counterstained with nuclear fast red (bars represent 100μm). <b>(F)</b> Immunohistochemistry for SPC (black), counterstained with nuclear fast red (bars represent 50μm).</p

Deletion of RhoA and RhoC together does not impair normal lung development.

<p>Rho^WT, RhoC^-/- and DKO mice were mated with CCSP-Cre mice on a K-Ras^WT background. <b>(A & B)</b> Lungs by H&E (bars represent 1mm and 200μm for panels A & B respectively). <b>(C)</b> Mice were crossed to a tdTomato to eGFP reporter line to assess for difference is Cre-expression pattern (bars represent 100μm). Green represents eGFP and Cre activity. Red presents tdTomato and the absence of Cre activity. Blue represents DAPI staining. <b>(D)</b> Immunohistochemistry for RhoA (brown) with hematoxylin counterstain (bars represent 25μm). <b>(E)</b> Immunohistochemistry for CCSP (black), counterstained with nuclear fast red (bars represent 100μm). <b>(F)</b> Immunohistochemistry for SPC (black), counterstained with nuclear fast red (bars represent 50μm).</p