Exon skipping induced by CRISPR-directed gene editing regulates the response to chemotherapy in non-small cell lung carcinoma cells.

We have been developing CRISPR-directed gene editing as an augmentative therapy for the treatment of non-small cell lung carcinoma (NSCLC) by genetic disruption of Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). NRF2 promotes tumor cell survival in response to therapeutic intervention and thus its disablement should restore or enhance effective drug action. Here, we report how NRF2 disruption leads to collateral damage in the form of CRISPR-mediated exon skipping. Heterogeneous populations of transcripts and truncated proteins produce a variable response to chemotherapy, dependent on which functional domain is missing. We identify and characterize predicted and unpredicted transcript populations and discover that several types of transcripts arise through exon skipping; wherein one or two NRF2 exons are missing. In one specific case, the presence or absence of a single nucleotide determines whether an exon is skipped or not by reorganizing Exonic Splicing Enhancers (ESEs). We isolate and characterize the diversity of clones induced by CRISPR activity in a NSCLC tumor cell population, a critical and often overlooked genetic byproduct of this exciting technology. Finally, gRNAs must be designed with care to avoid altering gene expression patterns that can account for variable responses to solid tumor therapy.

Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated nucleases (Cas) are among the most promising technologies for the treatment of hemoglobinopathies including Sickle Cell Disease (SCD). We are only beginning to identify the molecular variables that influence the specificity and the efficiency of CRISPR- directed gene editing, including the position of the cleavage site and the inherent variability among patient samples selected for CRISPR-directed gene editing. Here, we target the beta globin gene in human CD34+ cells to assess the impact of these two variables and find that both contribute to the global diversity of genetic outcomes. Our study demonstrates a unique genetic profile of indels that is generated based on where along the beta globin gene attempts are made to correct the SCD single base mutation. Interestingly, even within the same patient sample, the location of where along the beta globin gene the DNA is cut, HDR activity varies widely. Our data establish a framework upon which realistic protocols inform strategies for gene editing for SCD overcoming the practical hurdles that often impede clinical success.

MicroRNA Expression Profiling of Normal and Malignant Human Colonic Stem Cells Identifies miRNA92a as a Regulator of the LRIG1 Stem Cell Gene.

MicroRNAs (miRNAs) have a critical role in regulating stem cells (SCs) during development, and because aberrant expression of miRNAs occurs in various cancers, our goal was to determine if dysregulation of miRNAs is involved in the SC origin of colorectal cancer (CRC). We previously reported that aldehyde dehydrogenase (ALDH) is a marker for normal and malignant human colonic SCs and tracks SC overpopulation during colon tumorigenesis. MicroRNA expression was studied in ALDH-positive SCs from normal and malignant human colon tissues by Nanostring miRNA profiling. Our findings show that: (1) A unique miRNA signature distinguishes ALDH-positive CRC cells from ALDH-positive normal colonic epithelial cells, (2) Expression of four miRNAs (miRNA200c, miRNA92a, miRNA20a, miRNA93) are significantly altered in CRC SCs compared to normal colonic SCs, (3) miRNA92a expression is also upregulated in ALDH-positive HT29 CRC SCs as compared to ALDH-negative SCs, (4) miRNA92a targets the 3'UTR of LRIG1 SC gene, and (5) miRNA92a modulates proliferation of HT29 CRC cells. Thus, our findings indicate that overexpression of miRNA92a contributes to the SC origin of CRC. Strategies designed to modulate miRNA expression, such as miRNA92a, may provide ways to target malignant SCs and to develop more effective therapies against CRC.

Somatostatin signaling via SSTR1 contributes to the quiescence of colon cancer stem cells.

BACKGROUND: Neuroendocrine cells (NECs) reside adjacent to colonic stem cells (SCs) in the crypt stem cell (SC) niche, but how NECs are involved in regulation of SCs is unclear. We investigated NECs expressing somatostatin (SST) and somatostatin receptor type 1 (SSTR1) because SST inhibits intestinal proliferation. HYPOTHESIS: SSTR1 cells maintain SCs in a quiescent state, and aberrant SST signaling contributes to SC overpopulation in colorectal cancer (CRC). METHODS: The proportion of SCs to NECs cells was quantified, by flow cytometry, in CRC cell lines and primary normal/tumor tissues based on cellular ALDH and SSTR1 levels, respectively. Doubling time and sphere-formation was used to evaluate cell proliferation and stemness. CRC cell lines were treated with exogenous SST and SST inhibitor cyclosomatostatin (cycloSST) and analyzed for changes in SCs and growth rate. Paracrine signaling between NECs and SCs was ascertained using transwell cultures of ALDH+ and SSTR1+ cells. RESULTS: In CRC cell lines, the proportion of ALDH+ cells inversely correlates with proportion of SSTR1+ cells and with rate of proliferation and sphere-formation. While primary normal tissue shows SST and SSTR1 expression, CRC shows only SSTR1 expression. Moreover, ALDH+ cells did not show SST or SSTR1 expression. Exogenous SST suppressed proliferation but not ALDH+ population size or viability. Inhibition of SSTR1 signaling, via cycloSST treatment, decreased cell proliferation, ALDH+ cell population size and sphere-formation. When co-cultured with SSTR1+ cells, sphere-formation and cell proliferation of ALDH+ cells was inhibited. CONCLUSION: That each CRC cell line has a unique ALDH+/SSTR1+ ratio which correlates with its growth dynamics, suggests feedback mechanisms exist between SCs and NECs that contribute to regulation of SCs. The growth suppression by both SST and cycloSST treatments suggests that SST signaling modulates this feedback mechanism. The ability of SSTR1+ cells to decrease sphere formation and proliferation of ALDH+ cells in transwell cultures indicates that the ALDH subpopulation is regulated by SSTR1 via a paracrine mechanism. Since ALDH+ cells lack SST and SSTR1 expression, we conjecture that SST signaling controls the rate of NEC maturation as SCs mature along the NEC lineage, which contributes to quiescence of SCs and inhibition of proliferation.

Beyond the Genetic Code: A Tissue Code?

The genetic code determines how the precise amino acid sequence of proteins is specified by genomic information in cells. But what specifies the precise histologic organization of cells in plant and animal tissues is unclear. We now hypothesize that another code, the tissue code , exists at an even higher level of complexity which determines how tissue organization is dynamically maintained. Accordingly, we modeled spatial and temporal asymmetries of cell division and established that five simple mathematical laws ("the tissue code") convey a set of biological rules that maintain the specific organization and continuous self-renewal dynamics of cells in tissues. These laws might even help us understand wound healing, and how tissue disorganization leads to birth defects and tissue pathology like cancer.

APC mutations in human colon lead to decreased neuroendocrine maturation of ALDH+ stem cells that alters GLP-2 and SST feedback signaling: Clue to a link between WNT and retinoic acid signalling in colon cancer development.

APC mutations drive human colorectal cancer (CRC) development. A major contributing factor is colonic stem cell (SC) overpopulation. But, the mechanism has not been fully identified. A possible mechanism is the dysregulation of neuroendocrine cell (NEC) maturation by APC mutations because SCs and NECs both reside together in the colonic crypt SC niche where SCs mature into NECs. So, we hypothesized that sequential inactivation of APC alleles in human colonic crypts leads to progressively delayed maturation of SCs into NECs and overpopulation of SCs. Accordingly, we used quantitative immunohistochemical mapping to measure indices and proportions of SCs and NECs in human colon tissues (normal, adenomatous, malignant), which have different APC-zygosity states. In normal crypts, many cells staining for the colonic SC marker ALDH1 co-stained for chromogranin-A (CGA) and other NEC markers. In contrast, in APC-mutant tissues from familial adenomatous polyposis (FAP) patients, the proportion of ALDH+ SCs progressively increased while NECs markedly decreased. To explain how these cell populations change in FAP tissues, we used mathematical modelling to identify kinetic mechanisms. Computational analyses indicated that APC mutations lead to: 1) decreased maturation of ALDH+ SCs into progenitor NECs (not progenitor NECs into mature NECs); 2) diminished feedback signaling by mature NECs. Biological experiments using human CRC cell lines to test model predictions showed that mature GLP-2R+ and SSTR1+ NECs produce, via their signaling peptides, opposing effects on rates of NEC maturation via feedback regulation of progenitor NECs. However, decrease in this feedback signaling wouldn't explain the delayed maturation because both progenitor and mature NECs are depleted in CRCs. So the mechanism for delayed maturation must explain how APC mutation causes the ALDH+ SCs to remain immature. Given that ALDH is a key component of the retinoic acid (RA) signaling pathway, that other components of the RA pathway are selectively expressed in ALDH+ SCs, and that exogenous RA ligands can induce ALDH+ cancer SCs to mature into NECs, RA signaling must be attenuated in ALDH+ SCs in CRC. Thus, attenuation of RA signaling explains why ALDH+ SCs remain immature in APC mutant tissues. Since APC mutation causes increased WNT signaling in FAP and we found that sequential inactivation of APC in FAP patient tissues leads to progressively delayed maturation of colonic ALDH+ SCs, the hypothesis is developed that human CRC evolves due to an imbalance between WNT and RA signaling.

Increased Musashi-2 and Decreased NUMB Protein Levels Observed in Human Colorectal Cancer are reverted to Normal Levels by ATRA-Induced Cell Differentiation.

BACKGROUND: Musashi stem cell (SC) proteins (MSI-1 & MSI-2) are known to become over expressed during colorectal tumorigenesis in humans and mice. MSI-1 overexpression induces tumorigenesis through Notch activation via inactivation of NUMB. Previous studies also show that MSI-2 overexpression in mice induces intestinal tumorigenesis but the mechanism is independent of NUMB. However, whether the MSI-2/NUMB pathway contributes to colorectal cancer (CRC) development in humans is still undetermined. METHODS: We evaluated expression of MSI-2 and NUMB proteins in matched normal and CRC patient samples, as well as in human CRC cell lines. We also determined whether induction of cellular differentiation by all-trans retinoic acid (ATRA) influences MSI-2 and NUMB expression. RESULTS: Analysis of matched patient tissue samples and CRC cell lines showed that MSI-2 protein expression is significantly increased and NUMB expression is decreased in CRCs compared to the normal colonic tissue. Immunostaining of normal and adenomatous colonic epithelium revealed that MSI-1+ andMSI-2+ SCs reside in the SC niche and they become overpopulated during colon tumorigenesis. Moreover, promoting cellular differentiation by ATRA reduces MSI-2 protein levels, while increasing NUMB protein levels in human CRC cell lines. CONCLUSIONS: MSI-2/NUMB protein expression is altered during colon tumorigenesis, and indicates that MSI-2/NUMB signaling in human colonic stem cells is closely linked to normal colonic epithelial homeostasis. IMPLICATIONS: The ability to normalize MSI-2/NUMB signaling by inducing differentiation of cancer SCs suggests a novel therapeutic approach for CRC treatment.

Efficient Delivery and Nuclear Uptake Is Not Sufficient to Detect Gene Editing in CD34+ Cells Directed by a Ribonucleoprotein Complex.

CD34+ cells are prime targets for therapeutic strategies for gene editing, because modified progenitor cells have the capacity to differentiate through an erythropoietic lineage. Although experimental advances have been reported, the associated experimental protocols have largely been less than clear or robust. As such, we evaluated the relationships among cellular delivery; nuclear uptake, often viewed as the benchmark metric of successful gene editing; and single base repair. We took a combinatorial approach using single-stranded oligonucleotide and a CRISPR/Cas9 ribonucleoprotein to convert wild-type HBB into the sickle cell genotype by evaluating conditions for two common delivery strategies of gene editing tools into CD34+ cells. Confocal microscopy data show that the CRISPR/Cas9 ribonucleoprotein tends to accumulate at the outer membrane of the CD34+ cell nucleus when the Neon Transfection System is employed, while the ribonucleoproteins do pass into the cell nucleus when nucleofection is used. Despite the high efficiency of cellular transformation, and the traditional view of success in efficient nuclear uptake, neither delivery methodology enabled gene editing activity. Our results indicate that more stringent criteria must be established to facilitate the clinical translation and scientific robustness of gene editing for sickle cell disease.

The anti-cancer effect of retinoic acid signaling in CRC occurs via decreased growth of ALDH+ colon cancer stem cells and increased differentiation of stem cells.

BACKGROUND: Tumorigenesis is driven by stem cell (SC) overpopulation. Because ALDH is both a marker for SCs in many tissues and a key enzyme in retinoid acid (RA) signaling, we studied RA signaling in normal and malignant colonic SCs. HYPOTHESIS: RA signaling regulates growth and differentiation of ALDH+ colonic SCs; dysregulation of RA signaling contributes to SC overpopulation and colorectal cancer (CRC) development. METHODS: We analyzed normal and malignant colonic tissues and CRC cell lines to see if retinoid receptors (RXR & RAR) are exclusively expressed in ALDH+ SCs, and if RA signaling changes during CRC development. We determined whether RA signaling regulates cancer SC (CSC) proliferation, differentiation, sphere formation, and population size. RESULTS: RXR & RAR were expressed in ALDH+ colonic SCs, but not in MCM2+ proliferative cells. Western blotting/immunostaining of CRCs revealed that RA signaling components become overexpressed in parallel with ALDH overexpression, which coincides with the known overpopulation of ALDH+ SCs that occurs during, and drives, CRC development. Treatment of SCs with all-trans retinoic acid (ATRA) decreased proliferation, sphere formation and ALDH+ SC population size, and induced differentiation along the neuroendocrine cell (NEC) lineage. CONCLUSIONS: Retinoid signaling, by regulating ALDH+ colonic CSCs, decreases SC proliferation, sphere formation, and population size, and increases SC differentiation to NECs. Dysregulation of RA signaling in colonic SCs likely contributes to overpopulation of ALDH+ SCs and CRC growth. IMPLICATIONS: That retinoid receptors RXR and RAR are selectively expressed in ALDH+ SCs indicates RA signaling mainly occurs via ALDH+ SCs, which provides a mechanism to selectively target CSCs.

An miRNA Expression Signature for the Human Colonic Stem Cell Niche Distinguishes Malignant from Normal Epithelia.

Malignant transformation of tissue stem cells (SC) may be the root of most cancer. Accordingly, we identified miRNA expression patterns in the normal human colonic SC niche to understand how cancer stem cells (CSC) may arise. In profiling miRNA expression in SC-enriched crypt subsections isolated from fresh, normal surgical specimens, we identified 16 miRNAs that were differentially expressed in the crypt bottom, creating an SC signature for normal colonic epithelia (NCE). A parallel analysis of colorectal cancer tissues showed differential expression of 83 miRNAs relative to NCE. Within the 16 miRNA signature for the normal SC niche, we found that miR-206, miR-007-3, and miR-23b individually could distinguish colorectal cancer from NCE. Notably, miR-23b, which was increased in colorectal cancer, was predicted to target the SC-expressed G protein-coupled receptor LGR5. Cell biology investigations showed that miR-23b regulated CSC phenotypes globally at the level of proliferation, cell cycle, self-renewal, epithelial-mesenchymal transition, invasion, and resistance to the colorectal cancer chemotherapeutic agent 5-fluorouracil. In mechanistic experiments, we found that miR-23b decreased LGR5 expression and increased ALDH+ CSCs. CSC analyses confirmed that levels of LGR5 and miR-23b are inversely correlated in ALDH+ CSCs and that distinct subpopulations of LGR5+ and ALDH+ CSCs exist. Overall, our results define a critical function for miR-23b, which, by targeting LGR5, contributes to overpopulation of ALDH+ CSCs and colorectal cancer. Cancer Res; 77(14); 3778-90. ©2017 AACR.

The Proportion of ALDEFLUOR-Positive Cancer Stem Cells Changes with Cell Culture Density Due to the Expression of Different ALDH Isoforms.

A significant number of discrepancies exist within the literature regarding ALDEFLUOR-positive stem cell populations in cell lines. We hypothesized that these inconsistencies resulted from differences in culture conditions, particularly cell density. We cultured several colon cancer cell lines (N=8) at high and low densities and found a significant decrease in ALDEFLUOR-positive cell populations at high density. However, we found no changes in the CD166-positive stem cell population, self-renewal, or cell cycle distribution of cells cultured at different densities. Interestingly, when we sorted both ALDEFLUOR positive and negative populations from the different density cultures, we identified a significant number of Aldehyde dehydrogenase (ALDH) isoforms whose expression was decreased in ALDEFLUOR-positive stem cells cultured at high density. This novel finding suggests that multiple ALDH isoforms contribute to ALDEFLUOR activity in colon cancer stem cells and decreases in ALDEFLUOR-positive stem cells at high cell density are due to decreased expression of multiple ALDH isoforms. Thus, designing therapeutics to target ALDEFLUOR-positive cancer stem cells may require inhibition of multiple ALDH isoforms.