Studies conducted by two groups, including ours, were the first to show that aberrant activation of the Hh pathway occurs in the majority of pancreatic cancers (6
). Moreover, this ectopic activation seems to be due to Hh ligand overexpression rather than to activating mutations of genes involved in Hh signaling. This offers a unique opportunity to study pharmacologic Hh pathway inhibition by means of the small-molecule smoothened
inhibitor cyclopamine as a new therapeutic strategy for pancreatic cancer. Whereas blockade of Hh signaling causes growth inhibition of Hh-dependent pancreatic cancer cell lines in vitro
and a modest growth inhibition of the resulting s.c. pancreatic cancer xenografts in vivo
), the effects on pancreatic cancer metastases remain unexplored. To our knowledge, this is the first report specifically showing the potential for Hh inhibitors as a therapeutic option for limiting pancreatic cancer metastases. Thus, only one lung micrometastasis was observed in one of seven E3LZ10.7-bearing cyclopamine-treated mice, and complete absence of metastases was observed in xenografted mice treated with the combination therapy of cyclopamine and gemcitabine. Most notably, the striking reduction in pancreatic cancer metastases was present despite the apparent lack of significant effect of cyclopamine monotherapy on bulk primary tumor volume.
This abrogation of metastasis was also observed in xenografts derived from another pancreatic cancer cell line, L3.6pl, precluding this being only a special feature of one particular cell line.
For the first time, our study addresses two critical issues pertaining to the role of Hh pathway as a therapeutic target in pancreatic cancer. First, we show the significant correlation between extent of intracellular Hh activation and invasive ability, which is independent of any effects this signaling pathway may have on proliferation. Blockade of Hh signaling in pancreatic cancer cells significantly inhibits invasion, and, conversely, ectopic Hh activation in immortalized human pancreatic ductal cells renders them profoundly invasive in modified Boyden chamber assays. It has been previously reported that an active Hh pathway induces epithelial-to-mesenchymal transition through Gli1
-dependent transcriptional down-regulation of the cell adhesion molecule E-cadherin
), which, in turn, confers a greater invasive ability in cancer cells (20
). Our data confirm that similar mechanisms are active in the pancreatic epithelium, with profound loss of E-cadherin
expression in HPDE-Gli1
cells and the reciprocal up-regulation of E-cadherin
in E3LZ10.7 cells treated with cyclopamine.
The second issue that we address in this study pertains to minor cellular subpopulations that may be preferentially affected by Hh blockade in pancreatic cancer. Specifically, we show an ~3-fold reduction in the proportion of ALDH-expressing cells by cyclopamine therapy in E3LZ10.7 cells. Our finding of Gli overexpression in ALDH-bright cells might suggest that this subpopulation could be particularly Hh dependent and, therefore, preferentially sensitive to Hh inhibition with cyclopamine.
Expression of elevated ALDH levels in human hematopoietic progenitor cells was first reported more than 15 years ago; subsequently, the “ALDH-high” cells were shown to have significantly higher levels of engraftment when transplanted into nonobese diabetic/severe combined immunodeficient mice (17
). More recently, lineage-negative, ALDH-high cells isolated from acute leukemias were shown to have consistent ability to engraft in nonobese diabetic/severe combined immunodeficient mice, which is consistent with this subpopulation being enriched in leukemic stem cells (19
). The ~3-fold reduction of cells with elevated ALDH expression in treated E3LZ10.7 cells suggests that cyclopamine might be preferentially targeting the putative tumor-initiating subpopulation in pancreatic cancers, rather than the proliferative fraction comprising the bulk tumor volume.
How do we reconcile these two discrete effects of cyclopamine therapy, i.e., impairment of invasive capacity within the overall population of pancreatic cancer cells and a preferential depletion of a minor subpopulation of ALDH-expressing cells, in the context of observed in vivo
effects on metastasis inhibition? To address this question, one must consider the process of metastasis as one of tumor initiation occurring at a distant anatomic site. Given the numbers of cells released by a primary tumor into circulation per day (usually in millions) and the relatively few established metastases one encounters, the vast majority of disseminated cancer cells reaching potential metastatic sites are incapable of engraftment, and only a minority of circulating tumor cells retain the potential capacity to result in tumor initiation at secondary foci. Arguably then, a reduction in metastases can be observed with therapies that either limit the overall numbers of disseminated cells in circulation or with therapies that cause selective depletion of tumor-initiating cells even in the absence of discernible effects on cells that comprise the bulk of the tumor. For example, gemcitabine treatment significantly inhibited primary tumor growth in our series and concurrently reduced, but did not completely abrogate, the occurrence of macrometastases in the orthotopic xenograft model, which is consistent with previous reports (23
). Conventional antimetabolites such as gemcitabine likely reduce metastasis incidence by a sheer decrement in bulk tumor volume and proportional reduction in numbers of circulating tumor cells. In contrast, cyclopamine has a negative effect on metastatic seeding in pancreatic cancer that dramatically exceeds the observed efficacy on primary tumor volume, suggesting that cyclopamine either (a
) inhibits the functional ability of cancer cells to invade (e.g., by down-regulation of epithelial-to-mesenchymal transition) or (b
) preferentially targets the minor proportion of cells capable of tumor initiation within the bulk tumor without necessarily reducing overall invasive capacity, or (c
) results in a combination of (a
) and (b
). In contrast to gemcitabine, which unequivocally alters xenograft morphology, we find no significant histologic differences between control and cyclopamine-treated primary tumors, underscoring what we observe at the macroscopic level. Similarly, we find no differences in stromal neovasculature (as assessed by murine nestin immunohistochemistry and real-time PCR) between control and cyclopamine-treated arms, arguing against the possibility that reduced passive “leakage” of tumor cells into the circulation might account for the reduced frequency of metastases.
It is entirely possible that the lack of tumor growth inhibition in the orthotopic location is merely a pharmacokinetic issue that can be overcome with higher dosing. A previous report by Thayer et al. (5
), using s.c. xenograft models and delayed administration of cyclopamine (i.e., onset of cyclopamine treatment after s.c. xenografts became palpable), had shown a modest growth inhibition of 50% to 60% in two pancreatic cancer lines. Notably, even in this study, “concurrent” administration of cyclopamine (i.e., onset of cyclopamine treatment in conjunction with s.c. injection of cells) resulted in a more profound effect on xenograft growth inhibition (~80%), confirming that this class of agents has a greater effect on preventing tumor initiation than on established tumor regression. This finding is entirely compatible with our observed preferential effects on limiting metastases in the orthotopic model. Given a persistent requirement for Hh signaling in maintaining tissue homeostasis, one needs to be cognizant of potential toxicities that might arise from higher cyclopamine dosages in somatic Hh-dependent cell populations. Consistent with previous reports (reviewed in ref. 24
), we found no obvious signs of toxicity by Hh inhibition with cyclopamine at the given dose (25 mg/kg p.o. by oral gavage twice daily) in mice during the 30-day treatment period. It is possible that somatic stem cells, much like the bulk tumor cell population, are less Hh dependent than the circulating cancer cells that are destined to engraft at metastatic sites; however, this remains a matter of speculation.
We believe that our results provide a compelling rationale for exploring Hh inhibitors in human pancreatic cancer, particularly from the standpoint of therapy of metastatic disease. Despite small numbers, we show that the Hh transcription factor Gli1
is overexpressed at the mRNA level in four of eight samples from pancreatic cancer metastases as compared with matched primary tumor tissue, underscoring a role for this pathway in mediating disease progression. From an experimental therapeutics perspective, we also present an improvement in cyclopamine formulation that will facilitate its clinical translation. In previous preclinical reports, cyclopamine was usually administered dissolved in a triolein/ethanol or DMSO base, with adverse reactions (ulcerations) commonly seen at the injection site. In this study, we have used an orally bioavailable formulation of cyclopamine dissolved in cyclodextrin, which was administered by oral gavage at a twice-daily dosing schedule. Other orally bioavailable Hh small-molecule inhibitors have also been reported (25
) and, together with the current formulation or its analogues, are likely to be the used in future clinical trials.
In summary, we show for the first time that cyclopamine has a profound effect in limiting pancreatic cancer metastases in vivo. Our results potentially serve as the seedbed for a new paradigm in anticancer therapy, wherein a conventional antimetabolite that reduces bulk tumor volume (e.g., gemcitabine) is combined with a class of metastasis inhibitors like cyclopamine to enhance overall therapeutic efficacy and eventually ameliorate survival.