Therapeutic Trial of Metformin and Bortezomib in a Mouse Model of Tuberous Sclerosis Complex (TSC)

Tuberous sclerosis complex (TSC) is a human genetic disorder in which loss of either TSC1 or TSC2 leads to development of hamartoma lesions, which can progress and be life-threatening or fatal. The TSC1/TSC2 protein complex regulates the state of activation of mTORC1. Tsc2+/− mice develop renal cystadenoma lesions which grow progressively. Both bortezomib and metformin have been proposed as potential therapeutics in TSC. We examined the potential benefit of 1 month treatment with bortezomib, and 4 month treatment with metformin in Tsc2+/− mice. Results were compared to vehicle treatment and treatment with the mTORC1 inhibitor rapamycin for 1 month. We used a quantitative tumor volume measurement on stained paraffin sections to assess the effect of these drugs. The median tumor volume per kidney was decreased by 99% in mice treated with rapamycin (p = 0.0004). In contrast, the median tumor volume per kidney was not significantly reduced for either the bortezomib cohort or the metformin cohort. Biochemical studies confirmed that bortezomib and metformin had their expected pharmacodynamic effects. We conclude that neither bortezomib nor metformin has significant benefit in this native Tsc2+/− mouse model, which suggests limited benefit of these compounds in the treatment of TSC hamartomas and related lesions

Estradiol and mTORC2 cooperate to enhance prostaglandin biosynthesis and tumorigenesis in TSC2-deficient LAM cells

Lymphangioleiomyomatosis (LAM) is a progressive neoplastic disorder that leads to lung destruction and respiratory failure primarily in women. LAM is typically caused by tuberous sclerosis complex 2 (TSC2) mutations resulting in mTORC1 activation in proliferative smooth muscle–like cells in the lung. The female predominance of LAM suggests that estradiol contributes to disease development. Metabolomic profiling identified an estradiol-enhanced prostaglandin biosynthesis signature in Tsc2-deficient (TSC−) cells, both in vitro and in vivo. Estradiol increased the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis, which was also increased at baseline in TSC-deficient cells and was not affected by rapamycin treatment. However, both Torin 1 treatment and Rictor knockdown led to reduced COX-2 expression and phospho-Akt-S473. Prostaglandin production was also increased in TSC-deficient cells. In preclinical models, both Celecoxib and aspirin reduced tumor development. LAM patients had significantly higher serum prostaglandin levels than healthy women. 15-epi-lipoxin-A4 was identified in exhaled breath condensate from LAM subjects and was increased by aspirin treatment, indicative of functional COX-2 expression in the LAM airway. In vitro, 15-epi-lipoxin-A4 reduced the proliferation of LAM patient–derived cells in a dose-dependent manner. Targeting COX-2 and prostaglandin pathways may have therapeutic value in LAM and TSC-related diseases, and possibly in other conditions associated with mTOR hyperactivation

Renal cystadenoma histology in the treated mice.

<p>Representative tumor images are shown for each treatment cohort, selected from the kidneys with the largest tumor volume for each. Three cystadenoma each are shown at 100×, with a portion of the tumor indicated by the frame shown at 400× (below). Cystadenomas from control mice are shown in the first column, two from IP vehicle treated mice, and one from a sucrose treated mouse. Note the cystic nature of the tumors from the rapamycin-treated mice, as well as the flattened, thin nature of the cyst-lining cells, in contrast to cystadenomas from all other mice. Arrows point to prominent enlarged but viable cells in the bortezomib-treated mice.</p

Immunoblot analysis of kidney lysates to examine therapeutic specificity.

<p><i>Tsc2⁺⁻</i> A/J strain mice were treated with vehicle, rapamycin, bortezomib or metformin for 1 week, and kidney lysates were prepared for immunoblot analysis. A) Blot strips were incubated with the antibodies against pACC-S79, AKT, pS6-S235/236, pS6-S240/244 and S6. B) Blot strips incubated with antibodies against pRaptor-S792, GRP78 and AKT.</p

Macroscopic and microscopic kidney tumor scores for <i>Tsc2^+/−</i> mice treated with five different treatment regimens.

<p>Scores are shown for each kidney available from each mouse in these cohorts. A. Macroscopic kidney tumor scores. B–C. Microscopic kidney tumor volume is shown twice, on a linear y axis scale (B) and on a logarithmic y axis scale (C). For B and C, the number of mice and kidneys examined were: 5 and 7, 9 and 15, 7 and 12, 8 and 14, and 10 and 18, respectively, for IP vehicle, rapamycin, bortezomib, sucrose, and metformin cohorts, respectively.</p

Immunohistochemistry (IHC) analysis of therapeutic effects.

<p><i>Tsc2^+/−</i> A/J strain mice of ages 9–10 months were treated with drugs for 1 week, and kidneys prepared for histology. Sections were prepared and stained using pS6-S235/236 (red), pACC-S79 (red), pIκBα-S32/34 (red) and GRP78 (red) antibodies. The bottom panel shows apoptosis assessed by TUNEL method. Representative sections are shown. All images shown are taken at 100× magnification. Insets show portions of the tumor at higher magnification (400×).</p

Body weight of mice on treatment.

<p>Body weight of mice on treatment.</p