KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis
Goutham Narla, Analisa DiFeo, Yolanda Fernandez, Saravana Dhanasekaran, Fei Huang, Jaya Sangodkar, Eldad Hod, Devin Leake, Scott L. Friedman, Simon J. Hall, Arul M. Chinnaiyan, William L. Gerald, Mark A. Rubin, John A. Martignetti
Goutham Narla, Analisa DiFeo, Yolanda Fernandez, Saravana Dhanasekaran, Fei Huang, Jaya Sangodkar, Eldad Hod, Devin Leake, Scott L. Friedman, Simon J. Hall, Arul M. Chinnaiyan, William L. Gerald, Mark A. Rubin, John A. Martignetti
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Research Article Oncology

KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis

Abstract

Metastatic prostate cancer (PCa) is one of the leading causes of death from cancer in men. The molecular mechanisms underlying the transition from localized tumor to hormone-refractory metastatic PCa remain largely unknown, and their identification is key for predicting prognosis and targeted therapy. Here we demonstrated that increased expression of a splice variant of the Kruppel-like factor 6 (KLF6) tumor suppressor gene, known as KLF6-SV1, in tumors from men after prostatectomy predicted markedly poorer survival and disease recurrence profiles. Analysis of tumor samples revealed that KLF6-SV1 levels were specifically upregulated in hormone-refractory metastatic PCa. In 2 complementary mouse models of metastatic PCa, KLF6-SV1–overexpressing PCa cells were shown by in vivo and ex vivo bioluminescent imaging to metastasize more rapidly and to disseminate to lymph nodes, bone, and brain more often. Interestingly, while KLF6-SV1 overexpression increased metastasis, it did not affect localized tumor growth. KLF6-SV1 inhibition using RNAi induced spontaneous apoptosis in cultured PCa cell lines and suppressed tumor growth in mice. Together, these findings demonstrate that KLF6-SV1 expression levels in PCa tumors at the time of diagnosis can predict the metastatic behavior of the tumor; thus, KLF-SV1 may represent a novel therapeutic target.

Authors

Goutham Narla, Analisa DiFeo, Yolanda Fernandez, Saravana Dhanasekaran, Fei Huang, Jaya Sangodkar, Eldad Hod, Devin Leake, Scott L. Friedman, Simon J. Hall, Arul M. Chinnaiyan, William L. Gerald, Mark A. Rubin, John A. Martignetti

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Figure 2

Overexpression of KLF6-SV1 in PCa cell lines.

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Overexpression of KLF6-SV1 in PCa cell lines. (A) qRT-PCR analysis of pB...
(A) qRT-PCR analysis of pBABE and KLF6-SV1 vector–retrovirally infected PC3 and P3M cell lines demonstrated 12- and 27-fold overexpression, respectively, of KLF6-SV1 in pBABE-SV1 vector–infected cell lines compared with control. (B) KLF6-SV1–overexpressing cell lines proliferated significantly more than control cell lines, assessed by tritiated thymidine incorporation at 24 and 72 h of the PC3 and PC3M cell lines. Mean change in cell growth rate from 3 independent experiments is shown. (C) Increased expression of the oncogene c-myc and antiapoptotic Bcl-2, with concomitant reduction of the cyclin-dependent inhibitor p21, in KLF6-SV1–expressing cell lines. RNA and protein were harvested from 3 independent experiments, and qRT-PCR and Western blotting were performed. Overexpression of KLF6-SV1 increased Bcl-2 expression 50%, increased c-myc expression 80%, and reduced p21 expression 50% in PCa cell lines at the mRNA and protein levels. Actin was used as loading control for KLF6-SV1 and p21; tubulin was used for c-myc. (D) Increased invasion in KLF6-SV1 cell lines was associated with increased expression of MMP9. Gelatin zymography of the supernatant isolated from PC3 and PC3M cell lines is shown. Overexpression of KLF6-SV1 increased MMP9 expression in both PC3 and PC3M lines. KLF6-SV1–expressing stable PC3 cell lines (PC3-SV1) were 2-fold more invasive through a Matrigel basement membrane. The number of invasive cells was counted from 4 fields from 3 independent experiments. **P < 0.01, ***P < 0.001 versus control.