mTOR inhibition elicits a dramatic response in PI3K-dependent colon cancers.

The phosphatidylinositide-3-kinase (PI3K) signaling pathway is critical for multiple cellular functions including metabolism, proliferation, angiogenesis, and apoptosis, and is the most commonly altered pathway in human cancers. Recently, we developed a novel mouse model of colon cancer in which tumors are initiated by a dominant active PI3K (FC PIK3ca). The cancers in these mice are moderately differentiated invasive mucinous adenocarcinomas of the proximal colon that develop by 50 days of age. Interestingly, these cancers form without a benign intermediary or aberrant WNT signaling, indicating a non-canonical mechanism of tumorigenesis. Since these tumors are dependent upon the PI3K pathway, we investigated the potential for tumor response by the targeting of this pathway with rapamycin, an mTOR inhibitor. A cohort of FC PIK3ca mice were treated with rapamycin at a dose of 6 mg/kg/day or placebo for 14 days. FDG dual hybrid PET/CT imaging demonstrated a dramatic tumor response in the rapamycin arm and this was confirmed on necropsy. The tumor tissue remaining after treatment with rapamycin demonstrated increased pERK1/2 or persistent phosphorylated ribosomal protein S6 (pS6), indicating potential resistance mechanisms. This unique model will further our understanding of human disease and facilitate the development of therapeutics through pharmacologic screening and biomarker identification

Colon Cancer Tumorigenesis Initiated by the H1047R Mutant PI3K.

The phosphoinositide 3-kinase (PI3K) signaling pathway is critical for multiple important cellular functions, and is one of the most commonly altered pathways in human cancers. We previously developed a mouse model in which colon cancers were initiated by a dominant active PI3K p110-p85 fusion protein. In that model, well-differentiated mucinous adenocarcinomas developed within the colon and initiated through a non-canonical mechanism that is not dependent on WNT signaling. To assess the potential relevance of PI3K mutations in human cancers, we sought to determine if one of the common mutations in the human disease could also initiate similar colon cancers. Mice were generated expressing the Pik3caH1047R mutation, the analog of one of three human hotspot mutations in this gene. Mice expressing a constitutively active PI3K, as a result of this mutation, develop invasive adenocarcinomas strikingly similar to invasive adenocarcinomas found in human colon cancers. These tumors form without a polypoid intermediary and also lack nuclear CTNNB1 (β-catenin), indicating a non-canonical mechanism of tumor initiation mediated by the PI3K pathway. These cancers are sensitive to dual PI3K/mTOR inhibition indicating dependence on the PI3K pathway. The tumor tissue remaining after treatment demonstrated reduction in cellular proliferation and inhibition of PI3K signaling

Phospholipid ether analogs for the detection of colorectal tumors.

The treatment of localized colorectal cancer (CRC) depends on resection of the primary tumor with adequate margins and sufficient lymph node sampling. A novel imaging agent that accumulates in CRCs and the associated lymph nodes is needed. Cellectar Biosciences has developed a phospholipid ether analog platform that is both diagnostic and therapeutic. CLR1502 is a near-infrared fluorescent molecule, whereas 124/131I-CLR1404 is under clinical investigation as a PET tracer/therapeutic agent imaged by SPECT. We investigated the use of CLR1502 for the detection of intestinal cancers in a murine model and 131I-CLR1404 in a patient with metastatic CRC. Mice that develop multiple intestinal tumors ranging from adenomas to locally advanced adenocarcinomas were utilized. After 96 hours post CLR1502 injection, the intestinal tumors were analyzed using a Spectrum IVIS (Perkin Elmer) and a Fluobeam (Fluoptics). The intensity of the fluorescent signal was correlated with the histological characteristics for each tumor. Colon adenocarcinomas demonstrated increased accumulation of CLR1502 compared to non-invasive lesions (total radiant efficiency: 1.76×10(10) vs 3.27×10(9) respectively, p = 0.006). Metastatic mesenteric tumors and uninvolved lymph nodes were detected with CLR1502. In addition, SPECT imaging with 131I-CLR1404 was performed as part of a clinical trial in patients with advanced solid tumors. 131I-CLR1404 was shown to accumulate in metastatic tumors in a patient with colorectal adenocarcinoma. Together, these compounds might enhance our ability to properly resect CRCs through better localization of the primary tumor and improved lymph node identification as well as detect distant disease

Advanced Intestinal Cancers often Maintain a Multi-Ancestral Architecture.

A widely accepted paradigm in the field of cancer biology is that solid tumors are uni-ancestral being derived from a single founder and its descendants. However, data have been steadily accruing that indicate early tumors in mice and humans can have a multi-ancestral origin in which an initiated primogenitor facilitates the transformation of neighboring co-genitors. We developed a new mouse model that permits the determination of clonal architecture of intestinal tumors in vivo and ex vivo, have validated this model, and then used it to assess the clonal architecture of adenomas, intramucosal carcinomas, and invasive adenocarcinomas of the intestine. The percentage of multi-ancestral tumors did not significantly change as tumors progressed from adenomas with low-grade dysplasia [40/65 (62%)], to adenomas with high-grade dysplasia [21/37 (57%)], to intramucosal carcinomas [10/23 (43%]), to invasive adenocarcinomas [13/19 (68%)], indicating that the clone arising from the primogenitor continues to coexist with clones arising from co-genitors. Moreover, neoplastic cells from distinct clones within a multi-ancestral adenocarcinoma have even been observed to simultaneously invade into the underlying musculature [2/15 (13%)]. Thus, intratumoral heterogeneity arising early in tumor formation persists throughout tumorigenesis

Upstream activation of the PI3K pathway was unperturbed, but downstream signaling varied with response to rapamycin treatment.

<p>All <i>FC PIK3ca*</i> tumors and hyperplastic colon tissue demonstrated expression of p110*, the dominant active catalytic subunit of PI3K in this model. This resulted in increased phosphorylation of AKT⁹. The levels of pAKT and total AKT were not altered in response to rapamycin treatment. TSC2 total levels also did not vary. The levels of pmTOR, pSK1, and pS6, however, varied with rapamycin treatment. In tumors from the placebo cohort (−), high levels of pS6 were observed. In tumors that were treated with rapamycin (+), decreased levels of pS6 were seen in the majority of tumors and correlated with increased response on PET imaging. Increased pERK1/2 was observed in some placebo-treated cancers and also the rapamycin-treated tumor with persistent pS6 signaling. <i>FC PIK3ca*</i> tumor and hyperplastic tissue had increased cleaved caspase 3 due to increased cell turnover. A reduction in cleaved caspase 3 was identified in tumors possessing increased pERK1/2 signaling indicating that tumors with increased pERK may be resistant to rapamycin treatment through decreased apoptosis. GAPDH was used as a loading control.</p

Longitudinal monitoring with dual hybrid ¹⁸F-FDG PET/CT colonography revealed a dramatic tumor response in <i>FC PIK3ca*</i> mice treated with rapamycin.

<p>A group of 22 <i>FC PIK3ca*</i> mice were imaged with dual hybrid ¹⁸F-FDG PET/CT colonography and stratified based on tumor size. Mice were then treated with a placebo (ethanol dissolved in drinking water) or rapamycin 6 mg/kg/day by oral gavage for 14 days. Following completion of the treatment course, PET/CT colonography was repeated to evaluate for tumor response. In the mice receiving placebo, the tumors increased in size during the 14 day treatment period (<i>A, left</i>). In the rapamycin arm a significant response was noted (<i>A, right</i>). Projection and axial views are presented on the top and bottoms, respectively. The tumors were located on PET images and volumes were measured from CT scans data. The percent change in tumor volume for each tumor is displayed in a waterfall plot (<i>B</i>).</p

<i>Fc</i>^<i>+</i><i>Pik3ca</i>^{<i>H1047R</i>} colon cancers are similar to those in <i>Fc</i>^<i>+</i> <i>Pik3c</i>a^<i>p110*</i> mice.

<p>In both of these <i>Pik3ca</i> mutant models, deeply invasive cancers are seen with the vast majority of the tumors having penetrated below the muscularis mucosa. This is in contrast to <i>Apc</i>^<i>Min</i> colon tumors, which typically are adenomatous tumors with no or just superficial invasion. Abundant mucin is present within both the <i>Fc</i>^<i>+</i> <i>Pik3ca</i>^{<i>H1047R</i>} and <i>Fc</i>^<i>+</i> <i>Pik3c</i>a^<i>p110*</i> colon cancers. These <i>Pik3ca</i> mutant cancers also demonstrate increased proliferation, as measured by nuclear Ki67, in comparison to <i>Apc</i>^<i>Min</i> tumors. In addition, phosphorylated RPS6 and ERK1/2 are increased above that seen in the <i>Apc</i>^<i>Min</i> colon lesions. Scale bar for low magnification images = 1mm. Enlargements are 10x magnifications of the areas outlined in the low magnification images. Min, <i>Apc</i>^<i>Min</i>.</p