Hyperglycemia promotes K-Ras-induced lung tumorigenesis through BASCs amplification

Abstract

Oncogenic K-Ras represents the most common molecular change in human lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC). The presence of K-Ras mutation is associated with a poor prognosis, but no effective treatment strategies are available for K-Ras -mutant NSCLC. Epidemiological studies report higher lung cancer mortality rates in patients with type 2 diabetes. Here, we use a mouse model of K-Ras-mediated lung cancer on a background of chronic hyperglycemia to determine whether elevated circulating glycemic levels could influence oncogenic K-Ras-mediated tumor development. Inducible oncogenic K-Ras mouse model was treated with subtoxic doses of streptozotocin (STZ) to induce chronic hyperglycemia. We observed increased tumor mass and higher grade of malignancy in STZ treated diabetic mice analyzed at 4, 12 and 24 weeks, suggesting that oncogenic K-Ras increased lung tumorigenesis in hyperglycemic condition. This promoting effect is achieved by expansion of tumor-initiating lung bronchio-alveolar stem cells (BASCs) in bronchio-alveolar duct junction, indicating a role of hyperglycemia in the activity of K-Ras-transformed putative lung stem cells. Notably, after oncogene K-Ras activation, BASCs show upregulation of the glucose transporter (Glut1/Slc2a1), considered as an important player of the active control of tumor cell metabolism by oncogenic K-Ras. Our novel findings suggest that anti-hyperglycemic drugs, such as metformin, may act as therapeutic agent to restrict lung neoplasia promotion and progression.

Figure 1. Average blood glucose levels in WT and K-Ras^V12 mice before and after STZ treatment.

Tail-vein glucose was measured after 3 hours of fast. Mean ± standard error of mean; *p<0.05 versus untreated-K-Ras^V12; **p<0.05 versus untreated-WT (Student's t-test).

Figure 2. Histology and immunostaining of pancreas in treated and untreated mice with STZ. A.

(i, iv) H-E of a pancreatic islet in untreated and STZ-WT mice; (ii, v) and (iii,vi) reduction of Glut2 and insulin expression in STZ-WT mice compared to control mice. B. (i, iv) H–E of a pancreatic islet in untreated and STZ-K-Ras^V12 mice; (ii, v) and (iii,vi) reduction of Glut2 and insulin expression in STZ-K-Ras^V12 mice compared to untreated K-Ras^V12 control mice. Magnification 200×. Calibration bar: 50 µm.

Figure 3. Early and late time representation of tumor formation and progression, respectively.

A. Early time, after 4 weeks from STZ treatment, no tumor formation in untreated-K-Ras^V12 mice (i) and some tumors in K-Ras^V12 mice treated with STZ (ii). B. Late time, after 24 weeks from STZ treatment, well defined masses in untreated-K-Ras^V12 mice (i) and several multifocal tumor distribution in K-Ras^V12 treated with STZ (ii). C. Assessment of tumors at low and high grade in untreated-K-Ras^V12 and STZ- K-Ras^V12. Left: percent of low (white bar) and high (black bar) tumors in STZ- K-Ras^V12 and untreated-K-Ras^V12 mice (mean ± SEM; *, p<0.01 by X² test). Right: representative H-E of low and high lung tumor grade. H-E, magnification 100×. Calibration bar: 100 µm.

Figure 4. Assessment of tumor development and progression of lung tumors in STZ-K-Ras^V12 mice compared to untreated-K-Ras^V12 ones.

A. Average number of tumors that developed per mouse in STZ-K-Ras^V12 (black bar) compared to untreated-K-Ras^V12 (white bar) mice. B. Average tumor size (mm²) in STZ-K-Ras^V12 (black bar) and in untreated-K-Ras^V12 mice (white bar). C. Expression of neoplastic tissue with the tumor burden index which considers average number of tumors per mice and average size. Data are presented as mean ± SEM, *p<0.05 (Student's t-test).

Figure 5. Analysis of BASCs expansion.

A. Immunohistochemical analysis of CC10 and SP-C in terminal bronchioles of STZ-K-Ras^V12 (i, ii) and untreated-K-Ras^V12 (iii, iv) mice. Magnification 200×. Calibration bar: 50 µm. B. Bar graph indicates percentage of terminal bronchioles with 1 or more BASCs at early and late time (STZ-K-Ras^V12 vs STZ-WT: *p<0,01 by X² test).

Figure 6. Immunofluorescent staining of tissue sections to detect the Glut1 expression (red).

A. Terminal bronchioles of both STZ-K-Ras^V12 and untreated-K-Ras^V12 mice; B. Tumoral masses of STZ-K-Ras^V12 mice. Magnification 100×. Calibration bar: 100 µm.

Figure 7. Oncogenic K-Ras induces Glut1 expression in BASCs.

A. Immunofluorescent analysis of CC10 (green) and SP-C (red) dual positive BASCs of K-Ras (+/LSLG12Vgeo); RERTn (ert/ert) mice after 4-OHT treatment in vitro. B. Spheres from K-Ras (+/LSLG12Vgeo); RERTn (ert/ert) mice treated with 4-OHT in vitro show β-Gal staining and Glut1 expression compared with no Glut1 staining in control spheres from K-Ras (+/LSLG12Vgeo); RERTn (ert/ert) mice not treated with 4-OHT. Magnification 200×. Calibration bar: 50 µm.