In the past year, thanks to the support of the Cancer Research Foundation, we have made significant progress in our investigation of Let-7 microRNAs (miRNAs), a large family of genes that is believed to repress cancer. Our previous work, using mouse models and human cell culture models, has revealed that these miRNAs are critical for preventing colorectal cancer (CRC). When turned off (or turned down, which occurs in the majority of CRC tumors) their loss can fuel CRC progression. Since miRNAs target and repress other genes, we have worked toward identifying the key Let-7 targets that drive tumorigenesis, and thus, function as oncogenes.

Since Let-7 miRNAs are comprised of a relatively large gene family our first goal was to evaluate the individual and collective ability of these genes to repress tumor formation. To accomplish this we pursued new mutagenesis strategies for inactivating these genes in the mouse intestine, our model system. In proof-of-principle experiments where we attempted the inactivation of known tumor suppressors (whose loss should drive cancer), we are excited to report that this system is ready to proceed to the next phase of targeting Let-7 genes. This will hopefully illuminate the most important Let-7 gene(s), with the potential for future therapeutics designed to enhance their function.

Our second goal was to identify key targets that are repressed by Let-7, and determine how they may cooperate since their de-regulation (and elevation), in the context of Let-7 depletion, occurs in concert. We are excited to report our identification of a new Let-7 target, a transcription factor, a type of protein that acts to turn on, or turn off, specific target genes. We find that this transcription factor (or TF1 for short) is normally not present, or present at very low levels, in normal mouse and human intestine. However, from our analysis of published datasets and biopsies attained from Siteman Cancer Center we find that TF1 is elevated in both pre-cancerous lesions and CRC in a widespread fashion (in about two thirds of all tumors). Furthermore, when artificially turned on in non-malignant intestinal cells, in culture, that TF1 transforms cells to have a cancerous and a stem cell phenotype, which in cancer may augment tumor-initiating potential. Also, in every CRC cell line we have examined (n=6) TF1 is turned on, and when it is inactivated, CRC cell growth and movement is greatly reduced. Independent of Let-7, TF1 is amplified (increased copies of the gene) in about 10% of all CRC tumors. Through examination of TF1 targets, we have identified several growth factors and secreted proteins known to drive CRC progression. Interestingly, one of these growth factors, (GF1), is also turned on by two other Let-7 targets. Experiments in normal intestinal cells, in culture, reveal that TF1 and these other Let-7 targets cooperatively turn on GF1. GF1 is normally only found in the intestine at very low levels. This and other data supports our current hypothesis that Let-7 controls tumorigenesis by repressing factors that turn on GF1, a known driver of CRC. These findings are highly significant because of the widespread prevalence of TF1 in CRC tumors. Considering that TF1 is expressed at very low levels in normal human intestinal tissue, its pharmacological inhibition may cause few side effects if targeted for therapeutic purposes. This feature, and its widespread re-activation in CRC, may indicate that TF1 is an ideal “drugable” target.

In the past two years, thanks to the support of the Cancer Research Foundation, we have made significant progress in our investigation of Let-7 microRNAs (miRNAs), a large family of genes that is believed to repress cancer initiation and progression in multiple malignancies, including colorectal cancer, which is the focus of our research. Let-7 miRNA function is dependent on 12 unique genes in humans/mice, so in order to study the genetics and importance of these 12 genes, we endeavored to develop a “multiplexing” approach in live animals to simultaneously target multiple genes within single cells of the intestine. This model turned out to be hugely successful when we targeted known tumor suppressors (whose loss should drive cancer) in mouse intestine. Best of all, this model is completely controllable, with mutagenesis (to achieve gene inactivation) dependent upon administration of a drug, doxycycline. Mice given doxycycline in their chow developed aggressive intestinal cancers within 4-6 weeks. Examinations of mutations within these tumors revealed precise inactivation of the targeted genes, as expected. This model will be immensely useful for studying tumor formation driven by the inactivation of these four major tumor suppressors (Apc, Pten, Smad4, and P53). We can determine how these genes affect Let-7, and also how Let-7 inactivation fuels tumorigenesis in the context of these mutations. Additionally, we are now poised to embark on multiplex inactivation of Let-7 miRNAs, as well as other cancer-associated miRNA gene families. This will hopefully illuminate the most important Let-7 gene(s), whose enhancement may prove to be an effective method of therapy.

Our second goal was to identify key targets that are repressed by Let-7, and determine how they may cooperate since their de-regulation (and elevation), in the context of Let-7 depletion, occurs in concert. Integral to these studies, we discovered a new direct target of Let-7, called PLAGL2, a transcription factor that controls whether other genes are turned “on” or “off.” Several exciting relevant findings are as follows:

1)PLAGL2 drives normal cells to become cancerous, when turned on.

2)PLAGL2 inactivation in cancerous cells leads to a dramatic decrease in growth.

3)PLAGL2 inactivation in cancerous cells leads to a dramatic decrease in motility, a hallmark feature of metastatic cancers, that invade tissues to spread.

4)PLAGL2, when turned on, drives the formation of more stem cells, which are cells needed to give rise to all other cells in the intestine. This is key because PLAGL2 may also promote the formation of cells able to form new tumors.

5)PLAGL2 inactivation in normal intestinecells reduces the number of stem cells.

6)PLAGL2 inactivation does not affect growth of non-cancerous mini-“organoids” or miniature clumps of intestinal cells grown in culture as a model of normal intestine. This is key because PLAGL2 inhibition may have modest effects on normal tissuewhen attempting to inhibit PLAGL2 to slow/stop tumor growth.

7)PLAGL2 is very commonly activated in colorectal cancers (75% to 100%), and visibly turned on at all stages (early and metastatic) of disease.

8)PLAGL2 promotes downstream pathways already known to be involved/activated in colorectal cancer. This is consistent with PLAGL2 activation in the majority of colorectal cancers, and may, in fact, itself be integral to these major pathways.

9)PLAGL2 cooperates with another Let-7 target to activate the expression of a growth factor (IGF2) commonly up-regulated and known to be a driver of colorectal cancer.Therapies aimed at targeting IGF2 are in clinical trials.

These findings are very exciting and highly significant.We are currently developing mouse models in which we can turn “off” or turn “on” PLAGL2 in the intestine. This is key for studying PLAGL2 function in live animalsto determine how it drives cancer formation and progression. We are also developing cell culture models to determine what drugs and genes can control whether PLAGL2 is turned “on” or “off.”This will be very important for determining the underpinnings of aberrant PLAGL2 activation seen so commonly in colorectal cancer. Identifying drugs that can potentially turn “off” PLAGL2 will also be very important for potential therapeutic strategies in colorectal cancer.