Regulated Splicing Changes are Associated with Tumor Type and Grade

•Exon specific array from 3 lymphoma cell lines (IV, IVb and III) and non-tumor B cell line

•Colors vary from blue to red with increasing signal intensity and from dark to bright with the confidence on the value observed in multiple measurements

•Note the clustering of IV and IV samples

Relogio et al, JCB 2004

Tumor Cell Switch from Pyruvate Kinase M1 isoform to M2 isoform Promotes Tumor Growth

Christofk et al, Nature 2008Stable knockdown of PKM2 in thehuman lung cancer cell line H1299, rescued with M1 or M2 isoform

Tumor Cell Switch to Mena INV Isoform Promotes Metastasis

Goswami et al, Clin. Exp. Met. 2009

Philippar et al, Cancer Cell 2009

Expression of the INV isoform of Menapromotes metastasis

Invasive rat mammary adenocarcinoma linefollowing SCID mouse mammary xenograft

Global Analysis of Splicing Changes During EMT

RNA-seq analysis of splicing changes induced in HMLE-TwistER by Tam treatment

Shapiro et al Plos Genetics 2011

ESRP1 & ESRP2 are Key Regulators of Alternative Splicing During EMT

•Pulled out by a cDNA screen of 15,000 genes in 293T cells with a luciferase reporter of FGFR2-IIIb (epithelial) expression (293T cells normally express mesenchymal FGFR2-IIIc)

•Found that suppression of ESRP1 in PNT2 (human prostate cell line with epithelial expression profile) was sufficient to cause three characterized EMT splicing switches:

- CD44v8-10 to CD44s (epithelial to mesenchymal)- Mena 11a exclusion (epithelial to mesenchymal)- p120-catenin exon 2&3 inclusion (epithelial to mesenchymal)

Warzecha et al Mol. Cell. 2009

Knockdown of ESRP1 & ESRP2 Results in Mild Mesenchymal Phenotype

Lentiviral shRNAKnockdown in humanMammary epithelialCells (HMEC)

Warzecha et al EMBO 2010

Alternative Splicing of CD44

Ponta et al, Nature Reviews 2003

•CD44 binds hyaluronan•Mediates cell signaling as a platform and as a co-receptor

Reduced Inclusion of v6 in Some (but not all) Metastatic Cancers

Anti-CD44 v6 Anti-CD44 costitutive

Salmi et al, JCB 1993

benign cutaneous papilloma

squamocellular carcinoma

metastasis

Switch from CD44v to CD44s with EMT in vitro

A-C) HMLE/Twist-ER cellsD) HMLE/Snail-ER cells (left), TGF-β (5 ng/ml) treatment in HMLE cells (middle), orTwist expression in MDCK cells

Depletion of CD44 Inhibits EMT in vitro

A-C) HMLE/Twist-ER cells before and after of tamoxifen treatment (14 days A&B, 12 days C)D&E) HMLE cells before and after 18 days of TGF-β treatment (5 ng/ml )

EMT Can Be Rescued by CD44s not CD44v

B) HMLE/Twist-ER before (untreated) and after 12 days of TAM treatmentC) IF for E-cadherin at 12 daysD) MCF10A cells expressing CD44s or CD44v before and after 20 days of TGF-β (1 ng/ml)

ESRP1 Promotes Inclusion of v Domains and Inhibits EMT

A) HMLE/Twist-ER cells during TAM-induced EMTD) HMLE/Twist-ER cells before (untreated) and after 14 days of TAM treatmentE) HMLE before (untreated) and after 14 days of TGF-β treatment

A) MCF10A cells (spontaneously immortalized human breast epithelial line). MCF10AM is the same line induced to mesenchymal phenotype by TGF-beta. pAkt in with insulin stimulation.B) E-cadherin expression was blocked by PI3K inhibitor LY-294002C) Expression of CD44s, but not CD44v promoted pAkt D) Knockdown of CD44 inhibits insulin induced pAkt

CD44s (but not CD44v) Promotes Akt phosphorylation

E) CD44s but not CD44v expression blocks apoptosis in MCF10A cells in response to DNAcross-linker or growth in suspension F) Apoptosis-block in MCF10A cells expressing CD44s is reduced by inhibition of PI3K (LY-294002) but not MEK (U0126)

CD44s (but not CD44v) inhibits Apoptosis

A-C) Expression of CD44 isoforms in MMTV-rtTA/TetO-NeuNT mouse model in primary tumor and recurrent HER2/Neu-independent mesenchymal tumors that develop in the primary site after turning off HER2/Neu over-expression.

CD44v to CD44s Switch in Mesenchymal Mouse Tumor

A-C) Knockdown of CD44 in recurrent tumor derived cells inhibits incidence and burden. (5 × 105) were injected into the mammary fat pad of female FVB mice, harvested at 3 weeksD) Limiting (500 cells) injection at 5 weeks. shCD44 cells expressed CD44s, but not CD44vE&F) CD44 knockdown increases apoptosis to UV or cisplatin in these cells in vitro

CD44 Knockdown Inhibits Growth of Mesenchymal Transplant Tumor in Mouse

CD44s (not CD44v) RNA Expression is Correlated with Tumor Grade and N-cadherin Expression in Clinical Samples

•Both CD44v5/6 and CD44s were increase in tumor vs. normal tissue•CD44v5/6 was not significantly changed between tumor grade and did not correlate with N-cadherin

Conclusions & New Questions

•CD44v to s isoform switching occurs during EMT (known) and is functionally important in the Twist, Snail and TGFb induced EMT in human epithelial cells lines in vitro

•Isoform switching is correlated with mesenchymal phenotype in mouse mammary tumorin vivo and in human clinical samples

•Isoform switching is regulated in large part by ESRP1 (known)

•CD44s variant protects against apoptosis through Akt/Pi3k

•Is the EMT induced splicing switch conserved across tumor types? -Earlier work suggested that CD44v promotes metastasis (rat rat pancreatic

adenocarcinoma model) and that CD44v (not CD44s) is upregulated in brain mets

•What is the effect of the splicing switch on tumor metastasis?

Metastasis is Impaired in CD44 null Mice

Weber et al, Cancer Res. 2003

•Heterozygous p53 deficient mutants develop primary osteosarcomas•Analyzed liver and lungs (by serial sections) in 6 CD44+/+ and 4 CD44-/- mice