Although chemotherapeutic regimens consisting of 5-FU and other anticancer drugs, such as FOLFIRI or FOLFOX, are used as a first line of standard chemotherapy for patients with advanced colorectal cancer, virtually all the responses are incomplete and emergence of resistance, with subsequent recurrence of the cancer, is universal. Currently, effectiveness of conventional cancer therapeutics is usually evaluated by the reduction in tumor mass, resulting from elimination/killing of differentiated or undifferentiated cells that form the bulk of the tumor. However, the population of CSCs/CSLCs remains untouched and may even be enriched resulting in relapse of the disease. Earlier, we reported that exposure of colon cancer HCT-116 or HT-29 cells to FOLFOX that inhibited their growth led to enrichment of CSC/CSLC phenotype as evidenced by significantly increased proportion of CD44-, CD166-, and/or CD133-positive cells [8
] accompanied by increased colonosphere forming ability in vitro
and tumor formation in SCID mice [9
It has been well documented that dysregulation of Wnt/β-catenin signaling plays a pivotal role in the regulation of colonic stem cells, which has also been implicated in colon carcinogenesis [25
]. Our earlier studies have also shown that Wnt/β-catenin signaling pathway plays a pivotal role in regulating growth and maintenance of CSC/CSLC-enriched colonies derived from various human colon cancer cells such as colonospheres [20
]. The colonospheres are found to express LGR5, CD44, CD166, Musashi-1, and epithelial-specific antigen, which are markers of CSCs/CSLCs, and show elevated levels of total β-catenin causing exaggerated transcriptional activation of TCF/LEF gene responsible for the progression of cancer stem cells [20
]. Colon cancer HCT-116 cells are heterozygous for β-catenin, harboring one wild-type allele and one mutant allele with inactivation of SER45, one of the residues phosphorylated by GSK3β [24,26
]. Since one of alleles is wild type, we believe that this might be responsible for siRNA-induced down-regulation of β-catenin.
More interestingly, our studies also show that miR-21, whose levels are greatly increased in colon tumors, is also greatly elevated in CR colon cancer cells [9
]. Overexpression of miR-21 led to increased TCF/LEF activity, increased sphere forming ability in vitro
, and tumor formation in SCID mice and that down-regulation of miR-21 in CR colon cancer cells markedly decreased their sphere-forming ability, a phenomenon similar to that noted for β-catenin downregulated cells, supporting the contention that miR-21 regulates some of the functional properties of colon CSCs [9
These observations prompted us to examine whether down-regulation of miR-21 in CR colon cancer cells would induce differentiation of CSCs/CSLCs and render them susceptible to the conventional or nonconventional chemotherapeutic regimens. The basis for this investigation is that while CSCs/CSLCs are resistant to conventional chemotherapy, differentiated or differentiating cells that form the bulk of the tumor are sensitive to chemotherapy. In the current investigation, we have observed that down-regulation of miR-21 in CR HCT-116 cells, which are highly enriched in CSCs/CSLCs, leads to differentiation as evidenced by increased expression of gastrointestinal differentiation marker CK-20 [23
] and an increase in alkaline phosphatase activity [27,28
] and may also be causally related to decreased levels of CD44, pri-miR-21, and EGFR.
We have also hypothesized that down-regulation of miR-21 in CR cells that leads to differentiation would render them susceptible to conventional or nonconventional chemotherapeutics. CDF, a newly developed analog of curcumin with a greater bioavailability than the parent compound, have been shown to exert a greater growth inhibitory property than curcumin [7
]. Recent studies also showed that CDF was much more superior in the killing of gemcitabine-resistant pancreatic cancer cells with epithelial-to-mesenchymal phenotype that is reminiscent of CSCs [29
]. We have reported that CDF in combination with FUOX causes growth inhibition in CR colon cancer cells in vitro
, decreases the proportion of CSCs/CSLCs, and inhibits expression of miR-21 [7
]. Our current observation that following down-regulation of miR-21 in CR colon cancer HCT-116 cells they become highly susceptible to the growth inhibitory properties of FUOX, CDF, and CDF + FUOX clearly indicate increased vulnerability of CSC/CSLC-enriched CR colon cancer cells not only to conventional therapeutic FUOX but also to nonconventional therapeutics such as CDF or the combination of FUOX and CDF. Furthermore, the fact that downregulation of CK-20 by the corresponding siRNA produced no significant change in cellular growth of CR colon cancer cells in the absence or presence of FUOX suggest that CK-20 per se
is not involved in regulating the growth of CR colon cancer cells but acts as a marker of differentiation.
In conclusion, the results of our current investigation suggest that down-regulation of miR-21 is an effective therapeutic strategy for CR colon cancer by regulating cancer stem cell differentiation and subsequently rendering them susceptible to therapeutic agents.